Methods for measuring famin activity

Abstract

This invention relates to methods of measuring the activity of a FAMIN protein that comprise determining the aspartate transaminase activity of the FAMIN protein, for example by measuring the consumption of aspartate and / or α-ketoglutarate in the presence of the FAMIN protein and / or the production of oxaloacetate and / or glutamate. Methods of measuring and screening FAMIN activity are provided.

Description

[0001] Methods for Measuring FAMIN Activity

[0002] Field

[0003] The present invention relates to methods for measuring FAMIN activity and methods of screening for compounds that modulate FAMIN activity.

[0004] The enzyme FAMIN (also known as C13orf31 and Laccl) controls energy metabolism (7-3). Impaired or absent FAMIN activity reduces glycolytic flux, mitochondrial oxidative phosphorylation, ATP, ADP, AMP, and phosphocreatine levels; reduces cellular proton (H+) and lactate export; acidifies the cytoplasm and tips the cytoplasmic NADH:NAD+balance to a more reductive state. The latter is responsible for enhanced T cell priming by dendritic cells (3), mechanistically linking a surplus of reducing equivalents (i.e. electrons, e~) in the cytoplasm to strong genetic risk for autoimmune disease (Crohn’s and Still’s disease) elicited by hypomorphic (I254V) and loss-of-function (C284R) FAM IN variants (4, 5). How FAM IN exerts such fundamental control over energy metabolism has remained elusive.

[0005] FAMIN catalyses adenosine deaminase (adenosine + H2O ->• inosine + ammonia [NH3]), purine nucleoside phosphorylase (guanosine / inosine + orthophosphate [Pi] guanine / hypoxanthine + ribose-1 -phosphate [R1P]), and 5’-methylthioadenosine (MTA) phosphorylase (MTA + Pi adenine + methylthioribose-1-phosphate [MTR1P]) reactions (7). Additionally, FAM IN catalyses the direct interconversion of adenosine and adenine (adenosine + Pi adenine + R1P), which had previously been considered absent from eukaryotic metabolism. Only a few enzymes are known that catalyse at a single site chemically distinct reactions, and most are considered remnants of long-extinct earliest life (6). FAM IN has indeed closely related bacterial orthologues, including a protein of a thermophilic strain isolated from the hot springs of Yellowstone, and they all share FAMIN’s catalytic activities on these primordial metabolites (7, 7, 8).

[0006] The present inventors have unexpectedly found that the enzyme FAMIN (fatty acid metabolism-immunity nexus) displays aspartate transaminase activity. Assay methods and methods of screening for FAM IN modulators have been developed based on this finding.

[0007] A first aspect of the invention provides a method of measuring the activity of a FAMIN protein comprising;

[0008] providing a FAMIN protein; and

[0009] determining the aspartate transaminase activity of the FAMIN protein.

[0010] A second aspect of the invention provides a method of screening for a compound that modulates the activity of a FAMIN protein comprising;

[0011] determining the aspartate transaminase activity of a FAMIN protein in the presence and absence of a test compound,

[0012] wherein a difference in the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of test compound is indicative that the test compound modulates the activity of the FAMIN protein.In some embodiments, a method of the first or second aspect may comprise determining the aspartate transaminase activity of an isolated FAMIN protein. For example, aspartate transaminase activity may be determined by contacting the FAM IN protein with aspartate and a- ketoglutarate and measuring the consumption of aspartate or a- ketoglutarate or the production of oxaloacetate and glutamate. Alternatively, aspartate transaminase activity may be determined by contacting the FAM IN protein with oxaloacetate and glutamate and measuring the consumption of oxaloacetate or glutamate or measuring the production of aspartate or a-ketoglutarate. The aspartate, a-ketoglutarate, oxaloacetate or glutamate may be labelled. Suitable labelled substrates include isotopically labelled substrates.

[0013] In other embodiments, a method of the first or second aspect may comprise determining the aspartate transaminase activity of a FAMIN protein in a cell. For example, aspartate transaminase activity may be determined by contacting the cell with one or more labelled substrates of the forward or reverse aspartate transaminase reaction and measuring the amount of one or more labelled metabolites of the substrates in the cell. Preferably, aspartate transaminase activity may be determined by contacting the cell with labelled aspartate and measuring the amount of one or more labelled aspartate metabolites in the cell or contacting the cell with labelled glutamate and measuring the amount of one or more labelled glutamate metabolites in the cell. Suitable labelled substrates include isotopically labelled substrates.

[0014] Other aspects and embodiments of the invention are described in more detail below.

[0015] Brief Description of the Figures

[0016] Figure 1 shows that FAMIN is required for aspartate carbon flux, maintenance of mitochondrial membrane potential and cytoplasmic pH. (A) Schematic depiction of multi-catalytic FAMIN activities in context of the purine nucleotide cycle (PNC) and its relationship to the malate-aspartate shuttle, urea cycle, glycolysis, citrate- ma late shuttle, Krebs cycle, electron transport chain and glycerol-3-phosphate shuttle. Monofunctional enzymes sharing catalytic activities with FAMIN are also depicted. The new FAMIN activity reported herein is marked by dotted encircling. (B-K) Fractional incorporation in SW480WTand SW480FAMIN KOcells following a 3h pulse with 2 mM [13C415N] aspartate ± 10 pM GOT1 inhibitor GOT1-in-2c at 1* IC50 dose for partial blockade, or DMSO (vehicle).. (B) Total levels of succinyl-AMP (S-AMP) (n=5 / 6 control vs GOT1 i). (C, Left) Fractional incorporation of [15N], [13C4] and [13C415N] aspartate (n=5 / 6 control vs GOT1 i). (C, Right) Schematic depiction of [13C415N] aspartate stable isotope incorporation into depicted metabolites. (D-K) Fractional incorporation of [13C4] and [13C415N] S-AMP (D), [13C4] and [13C415N] argininosuccinate (E), [15N] glutamate (F), [13C4] citrate (G), isocitrate (H), fumarate (I) and malate (J) and [13C3] a-ketoglutarate (K) (n=5 / 6 control vs GOT1i). (L) Mitochondrial membrane potential, measured by JC10 fluorescence ratio, in SW480WT, SW480FAMIN KO1and SW480FAMIN KO2cells in response to Ctrl and 25 pM FCCP (n=8 control vs n= 4 FCCP). (M) Cytoplasmic pH, measured by BCECF fluorescence ratio, in SW480WT, SW48OFAMIN K01and SW48OFAMIN K02cells (n=8). (N-R) Total levels of IMP (N), hypoxanthine (O), inosine (P), guanine (Q) and guanosine (R) in SW480WTand SW480FAMIN KOcells following a 3 hr pulse of 2 mM [13C415N] aspartate ± 10 pM GOT1i (n=5 / 6 control vs GOT1i). Data represented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 and ****p <0.0001 (one-way ANOVA or two-way ANOVA as appropriate).

[0017] Figure 2 shows FAMIN-dependent cytoplasmic 5-carbon flux into the MAS and shift to cytoplasmic 4-carbons upon blockade of electron transfer to complex I. (A-G) Fractional incorporation in SW480WTand SW480FAMIN KOcells following a 3h pulse with 2 mM [13Cs15N] glutamate ± 100 pM GOT1 inhibitor GOT1-in-2c at10x IC50 dose for complete blockade, or DMSO (vehicle). (A, Left) Fractional incorporation of [13Cs] and [13Cs15N] glutamate (n=5 / 6 control vs GOT1i). (A, right) Schematic depiction of [13Cs15N] glutamate incorporation into aspartate and Krebs cycle intermediates. (B-D) Fractional incorporation of [13Cs] a-ketoglutarate (B), [15N], [13C4] and [13C415N] aspartate (C) and [13C4] citrate, isocitrate, succinate, fumarate and malate (D) (n=5 / 6 control vs GOT1i). (E) Total levels of aspartate (n=5 / 6 control vs GOT1i). (F-G) Fractional incorporation of [13C4] S-AMP (F) and [13C4] argininosuccinate (G) (n=5 / 6 control vs GOT1i). (H-Q) Fractional incorporation in SW480WTand SW480FAMIN KOcells following a 3h pulse with 2 mM [13C415N] aspartate ± 100 pM GOT1 inhibitor at 10* IC50 dose for complete blockade. (H, Left) Fractional incorporation of [13C4] and [13C415N] aspartate (n=5 / 6 control vs GOT1i). (H, Right) Schematic depiction of [13C415N] aspartate incorporation into glutamate, S-AMP, Krebs cycle intermediates and argininosuccinate. (I-K) Total levels of aspartate (I), S-AMP (J) and argininosuccinate (K) (n=5 / 6 control vs GOT1i). (L-Q) Fractional incorporation of [13C4] and [13C415N] S-AMP (L), [13C4] and [13C415N] argininosuccinate (M), [13C4] fumarate (N), malate (O), citrate (P), isocitrate (Q), [13C3] a-ketoglutarate and [13C2] succinate (Q) (n=5 / 6 control vs GOT1i). (R-Y) Fractional incorporation in SW480WTand SW480FAMIN KOcells following a 3h pulse with 2 mM [13C415N] aspartate ± 50 nM ETC complex I inhibitor IACS-010759 or DMSO (vehicle). (R, Left) Fractional incorporation of [13C4] and [13C415N] aspartate (n=4). (R, Right) Schematic depiction of [13C415N] aspartate incorporation into glutamate, S-AMP, Krebs cycle intermediates and argininosuccinate. (S) Total levels of aspartate (n=4). (T-U) Fractional incorporation of [13C4] S-AMP (T) and [13C4] argininosuccinate (U) (n=4). (V-W) Total levels of S-AMP (V) and argininosuccinate (W) (n=4). (X-Y) Fractional incorporation of [13C4] fumarate (X) and [13C4] malate (Y) (n=4). Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p <0.0001 (one-way ANOVA or two-way ANOVA, where appropriate).

[0018] Figure 3 shows that FAMIN is an aspartate transaminase. (A, top) Schematic depiction of the transaminase reaction tested using stable isotope-labelled substrates and their predicted products as indicated.13C in red,15N in blue. All reactions presented in this figure were performed using 1 pg recombinant protein, 50 pM each of the substrates, incubated for 3 hours at 37°C prior to quenching with an equal volume of 1% formic acid and metabolite detection by LC-MS. (A, bottom) Peak chromatographic areas of the reaction products [13C4] oxaloacetate and [15N] glutamate following incubation of FAMIN and appropriate controls including thiopurine methyltransferase (TPMT) with [13C415N] aspartate and a-ketoglutarate (representative of >10 independent experiments). (B) Representative chromatograms from (A) and (C), showing representative examples of peak intensities of the reaction products [13C4] oxaloacetate (m / z 135.0120), [15N] glutamate (m / z 147.0429), [15N] aspartate (m / z 133.0273) and [13Cs] a-ketoglutarate (m / z 150.0310) formed in the indicated reactions. (C, top) Schematic depiction of the ‘reverse’ transaminase reaction tested using stable isotope-labelled substrates and their products as indicated.13C in red,15N in blue. (C, bottom) Peak areas of the reaction products [13Cs] a-ketoglutarate and [15N] aspartate following incubation of FAMIN and appropriate controls including TPMT with [13Cs15N] glutamate and oxaloacetate (representative of >10 independent experiments). (D-E) Peak areas of the reaction products with recombinant FAMIN, catalytic-site mutant FAMINC284S, and appropriate controls. In (D) [15N] glutamate was detected from substrates [13C415N] aspartate and a-ketoglutarate, while in (E), [13Cs] a-ketoglutarate was detected from substrates [13Cs15N] glutamate and oxaloacetate (representative of 3 independent experiments). (F-G) Peak areas of reaction products following incubation of FAMIN, heat-denatured FAMIN, and appropriate controls. In (F) [15N] glutamate was detected from substrates [13C415N] aspartate and a-ketoglutarate, while in (G), [13Cs] a-ketoglutarate was detected from substrates [13Cs15N] glutamate and oxaloacetate (representative of 3 independent experiments). (H) Peak areas of [13Cs] glutamate following provision of FAMIN and appropriate controls with substrates [13Cs] a-ketoglutarate and alanine to assay foralanine transaminase activity. (I) Peak areas of [15N] glutamate following provision of YlmD and appropriate controls, including heat-denatured YlmD, with substrates [13C415N] aspartate and a-ketoglutarate (representative of 5 independent experiments). (J) Peak areas of [13Cs] a-ketoglutarate following provision of YlmD and appropriate controls, including heat-denatured YlmD, with substrates oxaloacetate and [13Cs15N] glutamate (representative of 5 independent experiments). (K) Peak areas of [13Cs] a-ketoglutarate following provision of FAMIN and appropriate controls, with substrates oxaloacetate and [13Cs15N] glutamate in the presence of 10 pM aminooxyacetic acid (AOA, a non-selective GOT inhibitor) or 10 pM GOT1 -selective inhibitor (GOT1-inh-2c) (representative of 2 independent experiments). (L) Peak Areas of [15N] glutamate following provision of FAM IN and appropriate controls with substrates [13C415N] aspartate and a-ketoglutarate in the presence of 10 pM AOA or 10 pM GOT1 -selective inhibitor.

[0019] Figure 4 shows that the PNC and FAMIN enable electro- meta bo lie oscillations in pancreatic islets (A) Schematic depiction of the purine nucleotide cycle (PNC), with relationship to glycolysis, and allosteric regulators of glycolysis’ pace-making enzyme phosphofructokinase- 1 (PFK-1; left panel). The three panels on the right side are reproduced from Tornheim & Lowenstein, J Biol Chem 1974, and highlight spontaneous, repeated oscillations in glycolytic and PNC metabolites (measured by spectrophotometry) in particle-free cytoplasmic extracts of rat skeletal muscle, sparked off by the provision of glucose and ATP at the start. AK, adenylate kinase; ADSL, adenylosuccinate lyase; ADSS, adenylosuccinate synthase; AMPD, AMP deaminase; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; HK, hexokinase; PFK-1, phosphofructokinase-1; GA3P, glyceraldehyde-3-phosphate; GAPDH, GA3P dehydrogenase; DHAP, di hydroxy ace tone phosphate; snG3P, glycerol-3-phosphate; GPDH, snG3P dehydrogenase; Pi, phosphate. (B-D) Representative [Ca2+]i oscillations in pancreatic islets isolated from (B) Famine2541(fully active), (C) Fam / np 254V(partially active) and (D) Famine284R(inactive) mice upon increasing glucose concentration in the perifusion medium from 2 to 11mM, and addition of the ADSS inhibitor L-alanosine. (E-G) Representative [Ca2+]i oscillations in pancreatic islets isolated from (E) Famine254', (F) Fam / np 254Vand (G) Fam / np 284Rmice upon increasing glucose concentrations in the perifusion medium from 2 to 11mM, and addition of the AMPD inhibitor Cpd3. (H) [Ca2+]i oscillations in a Fam / np 2541pancreatic islets as described in (B), in which discontinuing L-alanosine perifusion resulted in re-emergence of ‘slow’ oscillations. Data represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA or unpaired two-tailed Student’s t-test where appropriate)

[0020] Figure 5 shows that FAMIN affects total levels of argininosuccinate and Krebs cycle metabolites (A) Western blot of FAMIN in SW480WTand SW480FAMIN KOcells, p-actin, loading control. (B-H) Total levels of argininosuccinate (B), citrate (C), isocitrate (D), a-ketoglutarate (E), succinate (F), malate (G) and fumarate (H) in SW480WT, SW480FAMIN KOcells following a 3 hr pulse with 2mM [13C415N] aspartate ± 10 pM (1x|C5o) GOT1 inhibitor for partial inhibition (GOT1i) (n=5 / 6 for control vs GOTT). Data represented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 and ****p <0.0001 (one-way ANOVA or two-way ANOVA where appropriate).

[0021] Figure 6 shows the flux of glutamine, pyruvate and glucose into Krebs cycle metabolites (A-H) (Left) Fractional incorporation of [13C515N2] glutamine (A), [13Cs] a-ketoglutarate (B), [13C4] citrate (C), [13C4] isocitrate (D), [13C4] succinate (E), [13C4] fumarate (F), [13C4] malate (G), [13C4] aspartate (H), [13C4] S-AMP (I) and [13C4] argininosuccinate (J) in SW480WTand SW480FAMIN KOcells following a 3 hr pulse with 2 mM [13C515N2] glutamine (n=5) (Right) Schematic depiction showing [13Cs15N2] glutamine incorporation into Krebs cyclemetabolites. (K-Q) (Left) Fractional incorporation of [13Ca] pyruvate (K), [13C2] citrate (L), [13C2] isocitrate (M), [13C2] a-ketoglutarate (N), [13C2] succinate (O), [13C2] fumarate (P) and [13C2] malate (Q) in SW480WTand SW480FAMIN KOcells following a 3 hr pulse of 2 mM [13C3] pyruvate (n=5) (Right) Schematic depiction showing [13Ca] pyruvate incorporation into Krebs cycle metabolites. (R-X) (Left) Fractional incorporation of [13Ca] pyruvate (R), [13C2] citrate (S), [13C2] isocitrate (T), [13C2] a-ketoglutarate (U), [13C2] succinate (V), [13C2] fumarate (W) and [13C2] malate (X) in SW480WTand SW480FAMIN KOcells following a 3 hr pulse with 25 mM [13Ce] glucose pulse (n=5) (Right) Schematic depiction showing [13Ce] glucose incorporation into Krebs cycle metabolites. Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p <0.0001 (unpaired, two-tailed Student’s t test).

[0022] Figure 7 shows that complete GOT1 blockade affects total levels of Krebs cycle intermediates (A-F) Total levels of citrate (A), isocitrate (B), succinate (C), aKetoglutarate (D), fumarate (E) and malate (F) in SW480WT, SW480FAMIN KOcells following a 3 hr2 mM [13C415N] aspartate pulse ± 100 pM GOT1 inhibitor (GOT1i) (n=5 / 6 control vs GOTi). Data represented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 and ****p <0.0001 (one-way ANOVA or two-way ANOVA where appropriate).

[0023] Figure 8 shows that glutamate dehydrogenase inhibition does not affect [13C415N] aspartate tracing into Krebs cycle intermediates (A-G) Fractional incorporation of [13Cs] and [13Cs15N] glutamate (A), [13Cs] a-ketoglutarate (B), [13C4] citrate (C), [13C4] isocitrate (D), [13C4] succinate (E), [13C4] fumarate (F) and [13C4] malate (G) in SW480WTand SW480FAMIN KOcells following a 3 hr pulse of 2 mM [13Cs15N] glutamate ± 40 pM R162 glutamate dehydrogenase inhibitor R162 (GDHi) (n=5). Data represented as mean ± SEM. *p < 0.05, **p < 0.01 , ***p < 0.001 and ****p <0.0001 (one-way ANOVA or two-way ANOVA where appropriate).

[0024] Figure 9 shows that recombinant FAMIN and YlmD do not exhibit citrulline cleavage activity (A) Total levels of ornithine and citrulline in in SW480WT, SW480FAMIN KOcells. (B) Representative extracted chromatograms, using normalised peak intensity, of citrulline (left, m / z 176.1030); and ornithine (right, m / z 133.0971) following incubation of recombinant MBP-FAMIN, MBP-YImD or buffer control with 200 pM citrulline as indicated. The inset depicts the raw peak intensity of the chromatogram region corresponding to ornithine and shows the source fragmentation of citrulline to ornithine. (C) Representative extracted chromatograms showing raw peak intensities of citrulline (left, m / z 176.1030) and ornithine (right, m / z 133.0971) following incubation of Strepll-FAMIN in the presence of 100 pM citrulline as indicated. (D) Representative extracted chromatograms showing raw peak intensities of [13Cio15Ns] adenosine (left, m / z 283.1226) and [13Cs15Ns] adenine (right, m / z 146.0637) following incubation of recombinant MBP-FAMIN, MBP-YImD or buffer control (upper panels) or Strepll-FAMIN or buffer control (lower panels) or with 100 pM [13Cio15Ns] adenosine as indicated. Representative results of three replicates and multiple batches of recombinant proteins are depicted.

[0025] Figure 10 shows a proliferation defect in FAMIN-deficient tumour cells (A) Cell proliferation of SW480WT, SW480FAMIN KO1and SW480FAMIN KO2cells (Left), siCtrl and siFAMIN-silenced mouse melanoma B16F10 cells (Middle) and siCtrl and siFAMIN-silenced human breast carcinoma MCF7 (Right) cells 72h after siRNA transfection (n=8 SW480, n=10 B16F10, n=12 MCF7). (B-E) Fractional incorporation of [13C4] and [13C415N] aspartate (B), [13C4] citrate (C), [13C4] fumarate (D) and [13C4] malate (E) in siCtrl and siFAMIN-silenced B16F10 cells following a 3 hr pulse with 2mM [13C415N] aspartate (n=5). (F-N) Fractional incorporation of [13Cs] and [13Cs15N] glutamate (F), [13Cs] a-ketoglutarate (G), [13C4] succinate (H), [13C4] aspartate (I), [13C4] citrate (J), [13C4]isocitrate (K), [13C4] fumarate (L), [13C4] malate (M) and [13C4] and [13C415N] argininosuccinate (N) in siCtrl and siFAMIN-silenced B16F10 cells following a 3 hr pulse of 2mM [13Cs15N] glutamate (n=3). (O) Mitochondria Membrane Potential measurements in siCtrl and siFAMIN-silenced B16F10 cells in response to Ctrl and 25 pM FCCP treatment (n=9 control vs n=2 FCCP). (P) Cytoplasmic pH measurements in siCtrl and siFAMIN-silenced B16F10 cells (n=12). (Q-S) Cell proliferation of SW480WTand SW480FAMIN KOcells cultured with exogenous glutamate, aspartate, pyruvate (Q), inosine, guanosine, adenosine, methylthioadenosine (MTA) (R), hypoxanthine, guanine and adenine (S). Note cell culture media generally contained 2 mM glutamine. Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p <0.0001 (unpaired, two-tailed Student’s t test; one-way ANOVA or two-way ANOVA where appropriate).

[0026] Detailed Description

[0027] This invention relates to assay methods for measuring the activity of FAM IN protein by measuring the aspartate transaminase activity of the FAMIN protein and methods of identifying modulators of FAMIN protein by determining the effect of a test compound on aspartate transaminase activity.

[0028] FAMIN (fatty acid metabolism-immunity nexus) proteins are shown herein to display aspartate transaminase activity. In the forward direction, aspartate transaminase activity (also known as glutamate oxaloacetate transaminase activity) converts aspartate and a- ketoglutarate into oxaloacetate and glutamate. The amount of decrease of aspartate or a-ketoglutarate or the amount of increase of oxaloacetate or glutamate may be indicative of the amount of aspartate transaminase activity. In the reverse direction, aspartate transaminase activity converts oxaloacetate and glutamate into aspartate and a-ketoglutarate. The amount of decrease of oxaloacetate or glutamate or the amount of increase of aspartate or a-ketoglutarate may be indicative of the amount of aspartate transaminase activity in the reverse direction.

[0029] A FAMIN protein as described herein may be a eukaryotic FAMIN protein, preferably a mammalian FAMIN, such as human FAMIN. Human FAM IN (also known as LACC1 or C13orf3; Gene ID: 144811) may have the amino acid sequence of NP_001121775.1 (SEQ ID NO: 1) ora variant thereof. Human FAMIN may be encoded by the nucleotide sequence of NM_001128303.2 (SEQ ID NO: 2), NM_001350638.1, NM_001350639.1, NM_001350640.1 , NM_001350641.1 or NM_001350642.1 , or a variant thereof. In some embodiments, a FAMIN protein may comprise amino acids 176-430 (FAMINA176) of human FAMIN.

[0030] A FAMIN protein as described herein may comprise or consist of a DUF152 domain (Pfam02578, Cluster of Orthologous Group [COG] 1496). The DUF152 domain of a FAMIN protein may be identified using standard sequence analysis techniques. For example, the DUF152 domain is located in the C terminal portion (amino acids 176-430) of human FAMIN. The DUF152 domain may be responsible for the enzymatic activity of the FAMIN protein and the sequence of the DUF152 domain may be conserved between different FAMIN proteins.

[0031] In some embodiments, the FAMIN protein may be a variant FAMIN protein that has aspartate transaminase activity and comprises one or more amino acid substitutions, insertions or deletions relative to the reference sequence. For example, a variant FAMIN protein may comprise the amino acid sequence of

[0032] NP_001121775.1 (SEQ ID NO: 1) with an He residue ora Vai residue at a position corresponding to position254. In some preferred embodiments, a variant FAMIN protein may comprise a Cys residue at a position corresponding to position 284 of SEQ ID NO: 1.

[0033] In some embodiments, the aspartate transaminase activity of a FAMIN protein may be determined relative to an inactive FAMIN protein, such as a FAMIN protein with a Ser residue ora Arg residue at a position corresponding to position 284 of SEQ ID NO: 1

[0034] A FAMIN protein as described herein may be a prokaryotic FAMIN protein, preferably a bacterial FAMIN. Bacterial FAMIN proteins may include YlmD (Uniprot P84138) from Geobacillus stearothermophilus and YfiH (Uniprot P33644) from Escherichia coli strain K12.

[0035] In some embodiments, a method may comprise determining the effect of a test compound on the aspartate transaminase activity of a first FAMIN protein and a second FAMIN protein. A difference in effect of the compound on the aspartate transaminase activity of the first FAMIN protein relative to the second FAMIN protein may be indicative that the test compound is selective for the first FAM IN protein. For example, the first FAMIN protein may be human FAMIN protein and the second FAMIN protein may be a non-human FAMIN protein, such as a bacterial FAMIN protein.

[0036] A variant of a FAMIN protein or nucleotide sequence may share at least 50% sequence identity with the wildtype FAMIN amino acid or nucleotide sequence, for example the sequence of human FAMIN or FAMINA176, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. Particular amino acid sequence variants may differ from a wildtype FAMIN protein sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids

[0037] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147'. 195-197), the TBLASTN program, of Altschul et al. (1990) supra, or HMMER (S. R. Eddy. Current Opinion in Structural Biology, 6:361-365, 1996) generally employing default parameters. In particular, the psi-Blast algorithm may be used (Nucl. Acids Res. (1997) 253389-3402). Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

[0038] In some embodiments, the FAMIN protein may be an isolated FAMIN protein, for example a recombinant FAMIN protein.

[0039] An isolated FAM IN protein may be free or substantially free of contaminants or material with which it is naturally associated, such as other components of the cells or organelles with which it is found in its naturalenvironment, or the environment in which it is prepared (e.g. cell culture), when such preparation is by recombinant DNA technology practised in vitro or in vivo.

[0040] Isolated FAMIN protein as described herein may be provided using synthetic or recombinant techniques which are standard in the art. Alternatively, endogenous FAM IN protein may be purified from cells.

[0041] A recombinant FAMIN protein may be expressed in insoluble inclusion bodies in a prokaryotic expression system such as E coli and Lactococcus lactis. Following expression, the inclusion bodies may be isolated and solubilised with a denaturant to generate the FAMIN protein re-folded into soluble form. Alternatively, a recombinant FAM IN protein may be expressed in a eukaryotic expression system. Suitable eukaryotic host cells include mammalian cells such as CHO and CHO-derived cell lines (Lee cells), HeLa, COS, HEK293, HEK293T, and HEK-EBNA cells, amphibian cells such as Xenopus oocytes, insect cells, such as Trichoplusia ni, Sf9 and Sf21 and yeast cells, such as Pichia pastoris. In some preferred embodiments, recombinant FAMIN protein may be expressed in a mammalian cell line, such as HEK293T.

[0042] Recombinant techniques for the expression of proteins are standard in the art. The FAMIN protein may be coupled to a signal leader peptide to direct secretion of the fusion polypeptide from a eukaryotic cell into the culture medium. A range of suitable signal leader peptides are known in the art. The signal leader peptide may be a FAMIN signal sequence or may be heterologous to the FAMIN protein i.e. it may be a non-FAMIN signal sequence. For example, an a-factor secretion signal or BiP signal sequence may be employed.

[0043] Preferably, the signal peptide is removed by post-translational processing after expression of the FAMIN protein.

[0044] The expressed FAMIN protein may be isolated and / or purified, after production. This may be achieved using any convenient method known in the art. Techniques for the purification of recombinant polypeptides are well known in the art and include, for example HPLC, FPLC, size exclusion chromatography or affinity chromatography. In some embodiments, the expressed FAMIN protein may be partially purified before use in an assay method described herein.

[0045] In some embodiments, the FAMIN protein may be produced as a fusion protein further comprising an affinity tag, which may, for example, be useful for purification. An affinity tag is a heterologous peptide sequence which forms one member of a specific binding pair. Polypeptides containing the tag may be purified by the binding of the other member of the specific binding pair to the polypeptide, for example in an affinity column. For example, the tag sequence may form an epitope which is bound by an antibody molecule. Suitable affinity tags are well known in the art and are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533. The affinity tag sequence may be separated from the FAMIN protein after purification, for example, using a site-specific protease. In some preferred embodiments, recombinant human FAMIN protein with an affinity tag may be expressed in mammalian cells, such as HEK293T cells, and then purified by affinity chromatography followed by tag cleavage and size exclusion chromatography.

[0046] In other less preferred embodiments, the FAMIN protein may be comprised within a cell extract. The activity of one or more other metabolic enzymes with aspartate transaminase activity may be specifically inhibited in the cell extract . Suitable metabolic enzymes include GOT 1 or GOT2. The activity of the one or moremetabolic enzymes may be inhibited by any convenient means. Suitable techniques include genetic techniques, such as gene silencing, gene knockout, or gene knockdown, or chemical techniques, such as exposure to a selective or non-selective inhibitor. For example, GOT1 may be inhibited or partially inhibited by a selective inhibitor, such as GOT1-inh-2c [CAS No 732973-87-4]. Aspartate transaminase activity may also be determined in the cell extract relative to a control cell extract. A suitable control cell extract may be obtained from a cell in which FAM IN is inactivated. FAM IN may be inactivated in a cell by any convenient means, including genetic techniques, such as gene silencing, gene knockout, or gene knockdown, or chemical techniques, such as exposure to a selective or non-selective inhibitor.

[0047] The aspartate transaminase activity of a FAMIN protein may be determined in the assays described herein by contacting the FAMIN protein with aspartate and a-ketoglutarate and measuring the consumption of aspartate or a-ketoglutarate or the production of oxaloacetate or glutamate. For example, the aspartate transaminase activity of a FAMIN protein may be determined by contacting the FAMIN protein with labelled aspartate and / or labelled a-ketoglutarate, preferably with labelled aspartate, and measuring the production of labelled oxaloacetate and / or labelled glutamate. The labelled aspartate, labelled oxaloacetate, labelled a-ketoglutarate and / or labelled glutamate may be labelled with an isotope label (i.e. isotopically labelled). Suitable isotope labels include13C, and15N. In some preferred embodiments, the isotopically labelled aspartate may be [13C415N] aspartate, the isotopically labelled oxaloacetate may be [13C4] oxaloacetate, and the isotopically labelled glutamate may be [15N] glutamate (see Figure 3A).

[0048] The aspartate transaminase activity may also be determined in the assays described herein by contacting the FAM IN protein with oxaloacetate and glutamate and measuring the consumption of oxaloacetate or glutamate or the production of aspartate or a-ketoglutarate. For example, the aspartate transaminase activity of a FAMIN protein may be determined by contacting the FAMIN protein with labelled glutamate and / or labelled oxaloacetate, preferably labelled glutamate, and measuring the production of labelled aspartate and / or labelled a-ketoglutarate. The labelled glutamate, labelled aspartate, labelled oxaloacetate, and / or labelled a-ketoglutarate may be labelled with an isotope label (i.e. isotopically labelled). Suitable isotope labels include13C, and15N. In some preferred embodiments, the isotopically labelled glutamate may be [13C415N] glutamate, the isotopically labelled aspartate may be [15N] aspartate, and the isotopically labelled a-ketoglutarate may be [13Cs] a-ketoglutarate (see Figure 3C).

[0049] In some embodiments, the substrates in the methods described herein may be isolated molecules. Suitable molecules may be synthesised using conventional techniques or obtained from commercial suppliers (e.g. Sigma-Aldrich Corp, St. Louis, MO, USA). In other embodiments, one or more of the substrates in the methods described herein may be within a cell extract, for example an aqueous phase extract of cell metabolites). Suitable cell extracts may be devoid of protein and may include for example aqueous extracts obtained by Folch extraction (Folch et al., J Biol Chem 1957, 226, 497). An aqueous extract of substrates may be freeze-dried and re-suspended in phosphate-buffered saline.

[0050] The aspartate transaminase activity of a FAMIN protein may be determined under suitable reaction conditions. For example, the reactions may be performed in the presence of inorganic phosphate (Pi). In some embodiments, a phosphate containing buffer, such as PBS, may be used. The reactions may be performed at any convenient temperature, for example 32°C to 42°C, such as 37°C. The reactions may beperformed at any convenient pH, for example pH 6-8, such as pH 7.4. In some embodiments, aspartate transaminase activity may be determined in the absence of co-factors other than inorganic phosphate. In other embodiments, the activity of a FAMIN protein may be determined in the presence of a suitable cofactor, such as pyridoxalphosphate. Suitable reagents are available in the art (e.g. Aspartate Aminotransferase (AST) Activity Assay Kit (MAK055; Sigma Aldrich))

[0051] The consumption of a substrate or the production of a product in the methods described herein may be determined by any convenient technique. Suitable techniques include NMR, mass spectrometry, such as chromatography-coupled mass spectrometry (e,g, LC-MS), spectrophotometry, chemical assay methods and enzymatic assay methods. For example, the aspartate transaminase reaction may be coupled to a second reaction, such as a colorimetric reaction, to produce a detectable product detectable by spectrophotometry. In some preferred embodiments, the consumption of a substrate of the production of a product may be measured using liquid chromatography mass spectrometry (LC-MS).

[0052] In other embodiments, the FAMIN protein in the methods described herein may be within a cell. The cell may be a FAMIN proficient cell.

[0053] The aspartate transaminase activity in the cell may be determined by contacting the cell with one or more labelled substrates of the forward or reverse aspartate transaminase reaction and measuring the amount of one or more labelled metabolites of the substrates. The labelled substrate may be labelled with a detectable isotope, such as13C and / or15N .

[0054] In some embodiments, the one or more labelled substrates may be introduced to the cells in the form of a pulse. The concentration of labelled substrates in this pulse may be approximately equivalent to intracellular aspartate concentration. For example, the pulse of labelled aspartate may be 2 mM in concentration. The duration of the pulse may be any length sufficient to allow incorporation of labelled substrates. For example, the pulse may be 90 minutes, 3 hours, or 24 hours. Preferably, the pulse is 3 hours.

[0055] Preferably, aspartate transaminase activity may be determined by contacting the cell with labelled aspartate and measuring the amount of one or more labelled aspartate metabolites in the cell or contacting the cell with labelled glutamate and measuring the amount of one or more labelled glutamate metabolites in the cell.

[0056] The labelled aspartate may be labelled with an isotope, such as13C and / or15N. For example, the isotopically labelled aspartate may be [13C415N] aspartate.

[0057] The labelled aspartate metabolites may include labelled forms of aspartate, S-AMP, argininosuccinate, fumarate, malate, citrate, isocitrate, a-ketoglutarate, or succinate, for example forms labelled with an isotope (i.e. isotopomers). Suitable isotopically labelled aspartate metabolites may include [13CT5N] aspartate, [13C4] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, [13C4] citrate, [13C4] isocitrate, [13Ca] a-ketoglutarate, or [13C2] succinate. Preferably, the isotopically labelled aspartate metabolites may be [13C4] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, [13C4] citrate.The labelled glutamate may be labelled with an isotope, such as13C and / or15N. For example, the isotopically labelled glutamate may be [13Cs15N] glutamate.

[0058] The isotopically labelled glutamate metabolites include labelled forms of glutamate, a-ketoglutarate, aspartate, citrate, isocitrate, succinate, fumarate, malate, S-AMP, for example forms labelled with an isotope (i.e. isotopomers)> Suitable isotopically labelled glutamate metabolites may include [13Cs] glutamate, [13Cs15N] glutamate, [13Cs] a-ketoglutarate, [15N] aspartate, [13C4] aspartate, [13C415N] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, [13C4] citrate, [13C4] isocitrate, and [13C4] succinate,

[0059] The amount of one or more labelled substrate metabolites in a cell may be determined by any convenient technique. Suitable techniques include NMR, mass spectrometry, such as chromatography-coupled mass spectrometry (e,g, LC-MS), spectrophotometry, chemical assay methods and enzymatic assay methods. For example, the aspartate transaminase reaction may be coupled to a second reaction, such as a colorimetric reaction, to produce a detectable product detectable by spectrophotometry. In some preferred embodiments, the amount of labelled substrate or metabolite may be measured using liquid chromatography mass spectrometry (LC-MS).

[0060] In some embodiments, a method may further comprise determining aspartate transaminase activity as described herein a FAMIN-deficient cell. This may be useful for example in determining the background aspartate transaminase activity in a cell. Aspartate transaminase activity in the FAM IN proficient cell may be compared with aspartate transaminase activity in the FAMIN deficient cell to determine the aspartate transaminase activity that arises from the FAMIN protein.

[0061] A FAMIN-deficient cell is any cell in which FAMIN activity is reduced or abrogated compared to a wild-type FAMIN proficient cell. The FAMIN activity in a FAMIN-deficient cell may be reduced or abrogated by any convenient means, including genetic techniques, such as gene silencing, gene knockout, or gene knockdown, or chemical techniques, such as exposure to a selective or non-selective inhibitor. In some preferred embodiments, FAMIN-deficient cells may be generated using CRISPR / Cas9-mediated gene deletion. Suitable FAM IN deficient cells include cells expressing FAMIN with a mutation at C284, for example a C284S or C284R, mutation.

[0062] In some preferred embodiments, aspartate transaminase activity may be determined as described herein in mammalian cells, preferably human cells. Suitable cells include those useful for the modelling of disease, for example pancreatic p cell islets; murine melanoma cell lines, such as B16F10; human breast cancer cells lines, such as MCF-7; and human colorectal cancer cells, such as SW480 cells.

[0063] Aspartate transaminase activity may also be determined as described herein in primary cell lines, such as adipocytes, and organoids, such as intestinal organoids.

[0064] In some embodiments, the activity of one or more other metabolic enzymes with aspartate transaminase activity may be specifically inhibited in the cell. Suitable metabolic enzymes include GOT1 or GOT2. The activity of the one or more metabolic enzymes may be inhibited by any convenient means. Suitable techniques include genetic techniques, such as gene silencing, gene knockout, or gene knockdown, orchemical techniques, such as exposure to a selective or non-selective inhibitor. For example, GOT1 may be inhibited or partially inhibited by a selective inhibitor, such as GOT1-inh-2c [CAS No 732973-87-4].

[0065] Methods of measuring the aspartate transaminase activity of a FAMIN protein as described herein may be useful in screening for compounds that modulate, e.g. promote or inhibit, FAMIN activity.

[0066] For example, the aspartate transaminase activity of a FAMIN protein may be determined in the presence and absence of a test compound. A difference in the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of test compound is indicative that the test compound modulates the activity of the FAMIN protein. A decrease in activity in the presence of the test compound is indicative that the test compound is a FAM IN inhibitor. An increase in activity in the presence of the test compound is indicative that the test compound is a FAM IN potentiator.

[0067] In some embodiments, aspartate transaminase activity may be measured in a FAMIN proficient and a FAMIN deficient cell. A test compound that increases or reduces the difference between the aspartate transaminase activities in the FAMIN proficient cell and the FAMIN deficient cell may be identified as a compound that modulates the activity of the FAMIN protein.

[0068] A test compound that is determined to modulate the activity of the FAM IN protein by determining the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of the test compound may also be screened against other activities of the FAMIN protein, for example to identify specific modulators of the aspartate transaminase activity or general modulators of FAMIN activity. For example, the test compound may be screened against the adenosine deaminase, purine nucleoside phosphorylase, adenosine phosphorylase and methylthioadenosine phosphorylase activities of the FAMIN protein. Suitable activities are described in W02020 / 120406.

[0069] The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments. For example, in some embodiments, the amount of the above compounds may also be determined in a control in which FAM IN is inactivated.

[0070] A test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample. Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.

[0071] Test compounds may be consciously designed molecules, mixtures or extracts. For example, test compounds may be derivatives or analogues of compounds that are known to interact with FAM IN, such as the non-selective GOT inhibitor aminooxyacetate (AOA).Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate FAMIN activity. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances

[0072] The amount of test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used . Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100pM, e.g.

[0073] 0.1 to 50 pM, such as about 10 pM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development.

[0074] Suitable test compounds for screening include compounds that inhibit similar activities to the aspartate transaminase activity of FAMIN.

[0075] Suitable test compounds also include analogues, derivatives, variants and mimetics of any of the compounds listed above, for example compounds produced using rational drug design to provide test candidate compounds with particular molecular shape, size and charge characteristics suitable for modulating FAM IN activity.

[0076] A test compound identified as modulating FAMIN activity may be investigated further. For example, the selectivity of a compound for FAMIN may be determined by screening against other enzymes. Suitable methods for determining the effect of a compound on the activity of recombinant enzymes are well known in the art.

[0077] A test compound identified as modulating the aspartate transaminase activity of the FAMIN protein may be isolated and / or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and / or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application.

[0078] Following identification of a compound which modulates FAMIN activity as described herein, a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art.

[0079] Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

[0080] FAMIN modulating compounds may be useful in the treatment of diseases in animals, preferably humans. In particular, FAM IN modulating compounds may be useful in the treatment of cancer or metabolic diseases.Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.

[0081] It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0082] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

[0083] All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0084] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0085] Materials and Methods

[0086] Cell lines

[0087] The human cell lines SW480 (ATCC, Cat# CCL-228; RRID:CVCL_0546), HepG2 (ATCC, Cat# HB-8065; RRID:CVCL_0027), MCF7 (ECACC, Cat# 86012803; RRID:CVCL_0031) as well as the murine cell line B16-F10 (ATCC, Cat# CRL-6475; RRID:CVCL_0159) were maintained in complete DMEM (Gibco, Cat# 11500416) containing high glucose, with 2mM glutamine (Cat# 25030081) and 10% heat-inactivated FBS (Sigma Aldrich, Cat# F7524) without additional antibiotics in T75 cell culture flasks with vented caps (Corning, Cat# 353136) in a humidified incubator at 37°C and 5% CO2, unless indicated otherwise. Cells were split in a ratio of 1 :5 (HepG2, SW480, MCF7) or 1:10 (B16F10) every 3 days and discarded after a maximum of 20 passages. Regular testing for mycoplasma contamination was performed, and cell lines were regularly confirmed as mycoplasma-free.

[0088] Generation of knockout cell lines

[0089] All gRNAs used in this study were cloned into the backbone plasmid pLVPB-U6-sgRNAv2fl-shortccdB-PGK-Puro-BFP (a gift from Mathias Friedrich). This is an adaptation from a previously published construct (65), with an improved gRNA scaffold. The resulting constructs express U6-driven gRNAand PGK-driven Puromycin resistance gene, and Blue Fluorescent Protein (BFP). The gRNA sequences were designed using online tools provided by Synthego (https: / / www.synthego.com / products / bioinformatics / crispr-design-tool; human gene ID: ENST00000325686; murine gene ID: ENSMUST00000062789). Oligos were ordered from Sigma Aldrich, with overhangs compatible with Bbsl digestion of the backbone plasmid (for human cell lines: gRNA1: forward: CACCGagtcttcagtaatgtctgaGT, reverse: TTAAACtcagacattactgaagactC; gRNA2:

[0090] CACCGcttggccttggcagcatggGT, reverse: TTAAACccatgctgccaaggccaagC; for murine cell lines: gRNA1: forward: CACCGcagtcttgattgatctctcGT, reverse: TTAAACgagagatcaatcaagactgC, gRNA2: forward:CACCGaactgccatgagaccttacGT, reverse: TTAAACgtaaggtctcatggcagttC). For oligo annealing, sense and antisense oligos (10pM each) were mixed with T4 ligase buffer in 100pL reaction, incubated at 98°C for 5 minutes with a subsequent ramp down to 25°C at 5°C per minute. Annealed oligos (1:100 dilution) were then used for ligation to Bbsl-digested backbone plasmid using T4 DNA ligase (New England Biolabs, Cat# M0202S). Ligation products were transformed into Library Efficiency DH5a Competent Cells (ThermoFisher, Cat# 18263012) with recommended protocols. Single colonies were expanded, and plasmids extracted (PureYield Plasmid DNA Maxiprep, Promega, Cat# A2392). ForCas9 expression, pKLV2-EF1a-Cas9Bsd-W construct was used (66). This construct expresses EF1a-driven Cas9 followed by a T2A self-cleavage peptide and Blasticidin resistance gene. All cloned plasmids with gRNA sequences were verified by Sanger sequencing. FAMIN KO cell lines were generated using CRISPR / Cas9 technology, by transient co-transfection of gRNA and Cas9 expressing the respective constructs (detailed above). SW480, HepG2, and B16-F10 cells were reverse transfected using Lipofectamine 3000 as per the manufacturer’s instruction. Cells were then cultured with puromycin (1.5 - 3 g / mL) and Blasticidin S (10 pg / mL) for 3-4 days, for selection of cells co-expressing both gRNA and Cas9 constructs. To generate clonal cell lines, we then single-cell sorted these populations based on high BFP expression (NIHR Cambridge BRC Cell Phenotyping Hub). Sorted single cells were cultured in individual 96-wells in complete DMEM containing 1mM pyruvate, and 1X Pen / Strep antibiotics for one week. After one week, Pen / Strep was removed from the cell culture medium. Single colonies were expanded, genomic DNA extracted and indels confirmed by Sanger sequencing of the 1 kb region surrounding gRNA target site. Indels were confirmed by Synthego’s ICE analysis platform (https: / / ice.synthego.com / ). Finally, clones with 100% frame-shifting indels were expanded and knockout was confirmed using Western Blot for human cell lines, and ICE analysis for murine cell lines as no suitable antibody is commercially available. Clonal lines with confirmed knockouts were selected for further experiments.

[0091] Stable isotope tracing experiments

[0092] For stable isotope tracing experiments with [13C415N] aspartic acid (Sigma Aldrich, 607835), [13Cs15N] glutamic acid (Sigma Aldrich, 607851 ) [13Cs15N2] glutamine (Sigma Aldrich, Cat# 607983), [13Ce] glucose (Sigma Aldrich, 389374 ) and [13Ca] pyruvate (Sigma Aldrich, Cat# 490717), adherent cells in 6 well plates were washed once with warm PBS prior to incubation with medium containing the indicated concentration of labelled substrate for the indicated time, as stated in the figure legends. In experiments with [13C415N] aspartic acid and [13Ca] pyruvate, complete medium was used, whereas glucose-deficient medium (supplemented with FBS and glutamine) was used in [13Ce] glucose labelling experiments and glutamine-deficient medium (supplemented with FBS) was used in [13Cs15N] glutamic acid and [13Cs15N2] glutamine labelling experiments. Cells were treated with the indicated inhibitors in the tracing experiments shown in the figures, using DMSO as vehicle control. The inhibitors used were GOT1 inhibitor (Cambridge Bioscience, Cat# HY-122723), ETC inhibitor IACS-010759 (Cambridge Bioscience, Cat# 25867), and GDH inhibitor R162 (Cambridge Bioscience, Cat# HY-103096).

[0093] At the time of sample harvesting, plates were placed on ice, medium was removed and cells were washed two times with ice-cold PBS. Following aspiration of the PBS, 0.5 mL of ice-cold 4:1 methanol / water was added, cells were scraped and the suspension was transferred into 2 ml conical bottom screw-cap tubes. This was followed either by immediate extraction or storage at -80°C prior to extraction. Metabolites were extracted using a modified Folch method. In this way, 800 pL chloroform (Fisher Scientific, Cat# 15643700) and 380 pL milliQ water were added to samples, which were then vortexed, sonicated for 5 minutes andcentrifuged at 21,300 xg for 5 minutes. The top layer (aqueous phase) was transferred to Eppendorf tubes for drying using a SpeedVac vacuum centrifuge (Savant, Thermo Fisher Scientific, Cat# SC210A). Dried extracts were either analysed immediately or stored at -20°C for short-term storage, until resuspension and analysis by LC-MS (see ‘LC-MS analysis of aqueous metabolites’).

[0094] Plasmid cloning

[0095] The human FAMIN (wild-type) gene cloned in pESG-IBA105 vector containing a fusion of an N-terminal Twin-Strep-tag and TEV cleavage motif has previously been reported (1, 2). The mutant construct FAMINC284S was created by site-directed mutagenesis using the Q5® site-directed mutagenesis kit (New England Biolabs, Cat# E0554S) following the manufacturer’s instructions, and sequence-confirmed by Oxford Nanopore long-read sequencing technology (Plasmidsaurus). The YlmD gene cloned in pPSG-IBA105 vector containing a fusion of an N-terminal Twin-Strep-tag and TEV cleavage motif has previously been reported (1). The pET vector designed to express human thiopurine methyltransferase (TPMT) under the control of the T7 lac promoter was purchased from Vector Builder (Vector ID: VB221124-1237txg). A Strepll-tag was added by Gibson assembly to create the final construct. The MBP-FAMIN construct has previously been reported (1 ). The YlmD insert from Strepll-YImD was subcloned into a pMAL-c5X vector by Gibson assembly as per the manufacturer’s protocol (NEB Cat # E5510S) to generate the MBP-YImD plasmid.

[0096] Protein Purification

[0097] Strepll-YImD and Strepll-TPMT expression

[0098] Strepll-YImD was expressed in E. coli as previously described (1 ), and Strepll-TPMT followed the same protocol. Briefly, E. co# BL21 (DE3) cells transformed with the Strepll-YImD or TPMT expression plasmid were grown at 37°C in LB media containing 100 mg 1-1 ampicillin. Expression was induced at an GD600 of 0.6 with 1 mM IPTG for 18 hr at 16°C. Cultures were harvested by centrifugation at 5,000 g, frozen and then resuspended in lysis buffer containing 10 mM Tris, 20 mM NaCI, 10% glycerol, 2mM MgCI2, 1mM TCEP, pH 8.0, 1:10,000 (v / v) benzonase solution (Merck, Cat# E1014), 1 x complete Mini EDTA-free protease inhibitor cocktail tablet (Roche, Cat# 11836170001). The cells were lysed by sonication on ice. The lysate was clarified by centrifugation (30 min, 50,000 x g), incubated with Biolock (IBA Lifesciences, Cat# 15643608) (15 min, 4°C) and further clarified by centrifugation (15 min, 50,000 x g). The protein-containing supernatant was filtered through a 0.45 pm filter and loaded onto a 5 mL Strep-Tactin®XT 4Flow® column (IBA Lifesciences, Cat# 2-5033-001). The column was washed with 20 column volumes of 100 mM Tris, 200 mM NaCI, 10% glycerol, 2 mM TCEP, pH 8.0. The protein was eluted using 10 column volumes of 100 mM Tris, 200 mM NaCI, 10% glycerol, 2 mM TCEP, pH 8.0, 50mM biotin and eluted fractions were concentrated in a 10 kDa MWCO concentrator (Millipore Sigma Cat # UFC801096).

[0099] MBP-FAMIN and MBP-YImD

[0100] MBP-FAMIN was expressed in E. coli as previously described (1), and MBP-YImD followed the same protocol. Briefly, E. coli BL21(DE3) cells were transformed with MBP-FAMIN or MBP-YImD expression plasmids and grown at 37°C, 250 RPM, in ampicillin supplemented LB media. For MBP-FAMIN, expression was induced at an GD600 of 0.5 - 0.7 with 0.3 mM IPTG for 4 h at 30°C, 250 RPM. For MBP-YImD, expression was induced at an GD600 of 0.5 - 0.7 with 1.0 mM IPTG for 20 h at 16°C, 250 RPM. As previously described, cultures were harvested by centrifugation and cell pellets washed twice with PBS.Cells were then resuspended in 35 mL of lysis buffer containing 20 mM Tris-HCI, 200 mM NaCI, 1mM DTT, pH 7.4, 2* complete Mini EDTA-free protease inhibitor cocktail (Roche, Cat# 11836170001) and Benzonase (Merck, Cat# E1014). The cells were lysed by sonication on ice and cleared by centrifugation at 50,000* g for 40 minutes. The protein-containing supernatant was filtered through a 0.44 pm filter, and then loaded onto a pre-equilibrated dextrin sepharose MBPTrap High Performance column (Cytiva, Cat# 29-0486-41). The column was washed with 20 column volumes of lysis Wash buffer containing 20 mM Tris-HCI, 200 mM NaCI, 1mM DTT, pH 7.4. The protein was eluted with 10 column volumes of wash buffer containing 10 mM maltose. The eluted fractions were pooled and concentrated in a 10 kDa MWCO concentrator (Millipore Sigma Cat # UFC801096).

[0101] Human Strepll-FAMIN

[0102] Human Strepll-FAMIN was expressed in Expi293FTM cells and purified as previously reported (1). In brief, recombinant Strepll-tagged FAMIN (wild-type) and FAMINC284Swere expressed in Expi293FTM cells using the Expi293FTM Expression System Kit (GibcoTM, Thermo Fisher Scientific), following the manufacturer’s guidelines. 72 hours post transfection, the transfected cells were harvested by centrifugation (1 ,000g, 4°C), washed with ice-cold PBS and centrifuged at 1000g, 10 minutes, 4°C. The cell pellets were resuspended in hypotonic lysis buffer (10 mM HEPES, pH 8.0, 10 mM NaCI, 2 mM MgCI2, 10% glycerol) containing 1:10,000 benzonase® endonuclease (Merck, Cat# E1014) and cOmpleteTM Mini EDTA-free protease inhibitor cocktail (Roche Cat# 11836170001), lysed by sonication and clarified by centrifugation (50,000g, 20 minutes, 4°C). Post centrifugation, the supernatant was incubated with a biotin-blocking solution, BioLock (IBA Lifesciences, Cat# 15643608) on ice for 20 minutes. BioLock, containing the biotin-binding protein avidin, was added to block biotin and endogenous biotinylated proteins from subsequent binding to Strep-Tactin® resin without influencing the binding of the Twin-Strep tagged protein to the resin. After BioLock supplementation, the supernatant was further centrifuged (50,000g, 20 minutes, 4°C), sterile-filtered and applied to pre-equilibrated 5mL Strep-Tactin®XT 4Flow® column (IBA Lifesciences, Cat# 2-5033-001 ). The concentrated protein was further purified by sizeexclusion chromatography using a SuperdexTM 200 Increase 10 / 300 column (Cytiva Lifesciences, Cat# 28-9909-44) in buffer containing 100 mM HEPES, pH 7.6, 150mM NaCI. Fractions corresponding to recombinant FAMIN were collected, concentrated in a 10 kDa MWCO concentrator (Millipore Sigma), and assessed fortheir purities by silver-staining (Thermo ScientificTM Pierce® Silver Stain Kit, Cat# 24612) on a 4-15% SDS-polyacrylamide gel electrophoresis (Mini-PROTEAN® TGXTM Precast protein gels, BioRad, Cat #4561084) as previously described (1 ). The proteins were stored in -80°C for future use.

[0103] Catalytic assays with purified proteins

[0104] Enzymatic assay to assess the transamination activity

[0105] For catalytic assays to assess transamination, purified proteins were incubated with the following substrates as described in the figures: [13C415N] aspartate (Sigma Aldrich, Cat# 607835), [13Cs15N] glutamate (Sigma Aldrich, Cat# 60785), oxaloacetate (Sigma Aldrich, Cat# 04126), a-ketoglutarate (Sigma Aldrich, Cat# K1750). To assess for alanine transaminase activity [13Cs] a-ketoglutarate was used (MedChem Express, Cat HY-W013636S), alanine (Sigma Aldrich, A7627). Purified TPMT, after being confirmed to methylate its substrate 6-mercaptopurine, was used as a negative control for transaminase activity. Heat-denatured FAMIN and YlmD, inactivated by heating at 95°C for 5 minutes, were also used as negative controls. In addition to the GOT1 inhibitor used in cell experiments, the non-selective GOT inhibitor aminooxyacetate (AOA, Cambridge Bioscience Ltd, Cat# HY-107994-1mL) was used as an inhibitor in transamination reactions. Samples wereincubated for 3 hr at 37°C prior to quenching with 40 pl 1% formic acid and transferring to vials as above. Product formation was analysed by LC-MS using the C18-PFP methodology as below (see ‘LC-MS analysis of aqueous metabolites’).

[0106] Enzymatic assay to detect production of adenine, inosine and hypoxanthine

[0107] For mammalian expressed Strepll-tagged FAMIN, the reaction mixture (final volume of 40 pL) consisted of 0.48 pM (1 pg) of the protein and 10 pM of [13C1015N5] labelled adenosine as substrate (Cambridge Isotope Laboratories, Cat# CNLM-3806-CA) in Dulbecco’s PBS (pH 7.4) (Sigma, Cat# D8537)). For bacterially expressed MBP-FAMIN and MBP-YImD, the reaction mixture (final volume of 40 pL) consisted of 10 pM of the protein and 100 pM of [13Cio15Ns] labelled adenosine as substrate in Dulbecco’s PBS (pH 7.4) (Sigma, Cat# D8537). Mammalian expressed Strepll-FAMIN was incubated at 37 °C for 6 hours, followed by quenching with 40 pL of 1 % formic acid and subsequent centrifugation at 20,000 g for 10 minutes (23). Bacterially expressed fusion proteins were incubated at 37 °C for 1 hour, followed by quenching with 40 pL of 1 % formic acid. Similar reaction mixtures, namely, protein control, substrate control, and buffer control, were set up only with protein (absence of substrate), substrate (absence of protein) and protein elution buffer (absence of protein and substrate) respectively. As an internal standard, 10pL of 0.05 mg / pL aqueous extract from Algal lyophilized cells (U-13C, 98% (Cambridge Isotope Laboratories, Cat# CLM-2065-1) was added per sample. Adenosine, inosine, hypoxanthine and adenine were measured by LC-MS using the CSHC18 methodology below (see ‘LC-MS analysis of aqueous metabolites’).

[0108] Enzymatic assay to detect production of L-Ornithine

[0109] For mammalian expressed Strepll-tagged FAMIN, the reaction mixture (final volume of 40 pL) consisted of 0.48 pM (1 pg) of the protein, 100 pM L-citrulline substrate (Sigma, Cat# C7629), 1pM ZnSO4 (Merck, Cat# Z4750-500G) (23), in Dulbecco’s PBS (pH 7.4) (Sigma, Cat# D8537). For bacterially expressed MBP-FAMIN and MBP-YImD, the reaction mixture (final volume of 100 pL) consisted of 10 pM of the protein and 200 pM of L-citrulline as substrate, 1 pM ZnSO4 in Dulbecco’s PBS. Mammalian expressed Strepll-FAMIN was incubated at 37 °C for 6 hours, followed by quenching with 80 pL of ice-cold acetonitrile (23) and subsequent centrifugation at 20,000 g for 10 minutes (23). Bacterially expressed fusion proteins were incubated at 37 °C for 1 hour, followed by quenching with 200 pL of ice-cold acetonitrile and subsequent centrifugation at 20,000 g for 10 minutes (23). Similar reaction mixtures, namely, protein control, substrate control, and buffer control, were set up only with protein (absence of substrate), substrate (absence of protein) and protein elution buffer (absence of protein and substrate), respectively. The same Algal lyophilized cells U-13C internal standard was used in this assay.

[0110] Citrulline and ornithine were measured by LC-MS using the HILIC methodology below (see ‘LC-MS analysis of aqueous metabolites’).

[0111] LC-MS analysis of aqueous metabolites

[0112] Sample preparation

[0113] For samples from stable isotope tracing experiments, dried extracts were typically reconstituted in 100 pl of 10 mM ammonium acetate (Fisher Scientific, Cat# 10598410) in MilliQ water for downstream reversed phase C18-PFP analysis. Internal standards were omitted during isotopic labelling experiments to prevent contamination with labelled substrates. The samples were then vortexed and centrifugated to recover maximum volume. After centrifuging, the supernatants were transferred into SureSTART 0.3 ml vials (Fisher Scientific, Cat# 17324073) and capped with SureSTART 9 mm screw caps (Fisher Scientific, Cat # 17334063)ready for analysis. For HILIC analysis, dried extracts were reconstituted in 7:3 acetonitrile: water. For samples from catalytic assays, samples were transferred to 0.3 ml vials post-quenching and capped as above.

[0114] LC-MS methodologies

[0115] A Q Exactive Plus Orbitrap coupled to a Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system was used for all of the analysis (both Thermo Fisher Scientific). The majority of analysis, for example for the detection of nucleotides, nucleosides and TCA cycle intermediates, was carried out using an ACE Excel C18-PFP column (150 x 2.1 mm, 2.0 pm, Avantor, Cat# EXL-1010-1502U). Mobile phase A consisted of 0.1% formic acid (Fisher Scientific, Cat# 10596814) and 10 mM ammonium formate (Merck, Cat# 70221-25G-F) in MilliQ water and mobile phase B was acetonitrile (Fisher Scientific, Cat# 15624520) with 0.1% formic acid. For the analysis of TCA cycle intermediates, aspartate, glutamate and nucleotides, mobile phase A was 0.1% formic acid in MilliQ water. The column compartment temperature was 30°C, the flow rate was 0.5 mL / minute and the injection volume was 3.5 pL. The following gradient was used for compound elution: 0% B for 1.6 minutes, followed by an increase to 30% B over 2.4 minutes, a further increase to 90% B over 0.5 minutes and a hold at 90% B for 0.5 minutes, a return to 0% B over 0.1 minutes, with re-equilibration for 1.4 minutes, giving a total run time of 6.5 minutes. The needle wash used was 1:1 wateracetonitrile. Glutamine, nucleosides and nucleobases were analysed in positive ion mode MS whereas TCA cycle intermediates, aspartate, glutamate and nucleotides were analysed in negative ion mode MS. In positive ion mode, a full MS scan was performed with a scan range of 60.0 to 900.0 m / z and a resolution of 70,000. The sheath, auxiliary and sweep gas flow rates were 55, 15 and 3 arbitrary units, respectively, with a spray voltage of 3.5 kV, an S-lens radio frequency (RF) level of 50 and capillary and auxiliary gas heater temperatures of 275°C and 450°C, respectively. The source parameters in negative ion mode were identical, with the exception of a scan range of 55.0 to 825.0 m / z, a resolution of 140,000 and a spray voltage of 2.5 kV.

[0116] A hydrophilic interaction liquid chromatography (HILIC) approach, with a bridged ethylene hybrid (BEH) amide column (2.1x150 mm, 1.7 pm, Waters, Cat# 186009506), was used for the analysis of argininosuccinate, citrulline and ornithine. Mobile phase A was 100 mM ammonium carbonate (Merck, Cat # 1.59504.1000) in MilliQ water and mobile phase B was acetonitrile. The column compartment temperature was 30°C, the flow rate was 0.5 mL / minute and the sample injection volume was 5 pL. The following gradient was used for compound elution: 80% B for 1.5 minutes, followed by a decrease to 40% B over 3.5 minutes, a hold at 40% B for 1 minute, a return to 80% B over 0.1 minutes and re-equilibration for 3.9 minutes, for a total run time of 10 minutes. The needle wash used was 1:1 wateracetonitrile. Negative ion mode MS was used for analysis of argininosuccinate whereas positive ion mode MS was used for analysis of citrulline and ornithine. In positive ion mode, a full MS scan was performed with a scan range of 60.0 to 900.0 m / z and a resolution of 70,000. The sheath, auxiliary and sweep gas flow rates were 53, 14 and 3 arbitrary units, respectively, with a spray voltage of 3.5 kV, an S-lens RF level of 50 and capillary and auxiliary gas heater temperatures of 300°C and 438°C, respectively. The source parameters in negative ion mode were identical, with the exception of a spray voltage of 2.5 kV.

[0117] An ACQUITY Premier CSH C18 Column with VanGuard FIT (1.7 pm, 2.1 x 50 mm, Waters, Cat# 186009463) was used for the measurement of adenine and adenosine in catalytic assays. Mobile phase A was 10 mM ammonium formate with 0.1% formic acid in MilliQ water, and mobile phase B was acetonitrile with 0.1% formic acid. The column compartment temperature was 30°C, the flow rate was 0.8 ml / min, andthe sample injection volume was 2 pL. The following gradient was used for compound elution: 0% B for 0.5 minutes, followed by an increase to 20% B over 0.7 minutes, a further increase to 90% B over 0.1 minutes, a hold at 90% B for 0.3 minutes, a return to 0% B over 0.1 minutes and re-equilibration for 0.5 minutes, for a total run time was 2.2 minutes. The needle wash used was 1:1 watenacetonitrile. A full MS scan was performed in positive ion mode, with a scan range of 50.0 to 750.0 m / z and a resolution of 35,000. The sheath, auxiliary and sweep gas flow rates were 60, 18 and 4 arbitrary units, respectively, with a spray voltage of 3.5 kV, an S-lens RF level of 50 and capillary and auxiliary gas heater temperatures of 288°C and 475°C, respectively.

[0118] LC-MS data processing

[0119] Targeted data processing was carried out using Xcalibur (Version 4.1.31.9, Thermo Fisher Scientific, Cat# OPTON-30382). In labelling experiments, fractional incorporation (%) was defined as the peak area of the mass isotopomer of interest divided by the total peak areas of all mass isotopomers, expressed as a percentage. Whenever possible, area ratios were calculated by dividing the peak area of interest by the peak area of an isotopically labelled internal standard. To confirm identification, compound retention times were validated against known external standard solutions.

[0120] pHc measurements using BCECF-AM

[0121] Cytoplasmic pH (pHc) was measured by following the manufacturer’s instructions (Thermo Fisher, Cat# B1150). For cell line experiments, cells were seeded in a black 96-well plate with transparent bottoms (Greiner Bio-One, Cat# 655096) and silenced / treated as indicated. At the respective time point, cells were gently washed twice in warm Hank’s Balanced Salt Solution (HBSS) and incubated with 5 pM BCECF-AM in HBSS in a non-CO2 incubator for 30 minutes. Following this, wells were washed twice again with warm HBSS, and pHc was measured immediately on a CLARIOstar Plus plate reader (BMG Labtech) with dual excitation set at 490 nm and 440 nm and fixed emission at 535 nm.

[0122] For organoids, an adapted method was applied. In brief, small intestinal organoids were passaged once before use to increase purity and seeded in 50pL growth-factor reduced matrigel (Corning, Cat# 354230) and cultured in IntestiCult Organoid Growth Medium (Stem Cell Technologies, Cat# 06005). After 3-4 days, media was removed, organoids were harvested with one wash of cold phosphate-free PBS and pelleted. Organoids were then resuspended in 100 pL of TrypLE Express (ThermoFisher, Cat# 12605036) and incubated at 37°C and 5% CO2 for 4 minutes. Following digestion, single cells were washed once in DMEM and incubated with 1 pM BCECF in DMEM for 20 minutes. After incubation, single cells were washed once and technical triplicates per organoid were resuspended in FACS buffer with SYTOXTM Blue Dead Cell Stain (Thermo Fisher Scientific, Cat# S34857), transferred to FACS tubes and kept on ice protected from light until data acquisition on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

[0123] Mitochondria membrane potential

[0124] Mitochondrial membrane potential (MMP) was measured using the JC-10 Assay for Microplate Readers (Sigma-Aldrich, Cat# MAK159), following the manufacturer’s protocol. Cells were seeded in black-walled 96-well plates with clear bottoms (Greiner Bio-One, Cat# 655096) treated as indicated, and incubated with JC-10 Dye Loading Solution for 45 minutes at 37°C. JC-10 Dye Loading Solution was prepared by diluting 50 pL of 100* JC-10 in 5mL Assay Buffer A (MAK159B) and mixing thoroughly. Following incubation, Assay Buffer B (MAK159C) was added, and fluorescence was measured immediately using a CLARIOstar Plus plate reader (BMG Labtech) with dual excitation set at Aex = 490 nm / Aem = 525 nm and Aex = 540 nm / Aem = 590 nm. The red / green fluorescence ratio was used to assess MMP. FCCP (Sigma-Aldrich, Cat# C2920) was used as a positive control. Cells were protected from light throughout the assay.

[0125] Cell line proliferation assays

[0126] Cell proliferation was assessed using the CyQUANT Direct Cell Proliferation Assay (Thermo Fisher, Cat# C35011) following the manufacturer’s instructions. Cells were plated in clear flat- bottom 96-well plates (Greiner Bio-One, Cat# 655096). CyQUANT Direct 2X Detection Reagent was mixed thoroughly, allowed to stand at room temperature, then added to cells (1:1 ratio with culture medium) and incubated for 60 minutes at 37°C. Fluorescence intensity was measured using a CLARIOstar Plus plate reader (BMG Labtech) with 480 nm excitation and 535 nm emission. Treatments, including rescue agents (adenosine, Cat# A9251; CAS:58-61-7; inosine, Cat# I4625; CAS:58-63-9; guanosine, Cat# G6752, CAS:118-00-3; adenine, Cat# A8626, CAS: 73-24-5, hypoxanthine, Cat# H9377, CAS: 68-94-0; guanine, Cat# G11950, CAS73-40-5; sodium pyruvate, Cat# P5280, glutamine Cat# 25030081 , pyruvate) were added at the time of seeding, and cells were exposed to treatments for the duration of the experiment, unless indicated otherwise.

[0127] Transient silencing using small interfering RNA (siRNA)

[0128] For transient silencing, reverse transfection using Lipofectamine RNAiMax (ThermoFisher, Cat# 13778150) was performed as per manufacturer’s instructions. In brief, cells were trypsinized, spun at 300xg for 3 minutes, resuspended in complete DMEM and counted (Countess, ThermoFisher). Cells were only used when viability was >95%. For proliferation assays, 10 pL of final Lipofectamine RNAiMax / siRNA mix was added per well in a clearbottom 96-well plate (Greiner BioOne, Cat# 655096). After incubation for 5 minutes, 90pL of cell suspension containing 5,000 to 7,000 cells were added, and plates were incubated for 72-96 hours, as indicated. For silencing, ON-TARGET plus silencing RNAs, purchased from Horizon Discovery LTD were used (ON-TARGETplus Non-targeting Pool, Cat# D-001810-10; SMARTpool: ON-TARGETplus Lacd siRNA, Cat# L-052159-01; ON-TARGETplus Human LACC1 siRNA; Cat# L-015653-02). Silencing efficiency was tested using qPCR and was >90%. In brief, total cellular RNA was isolated (Qiagen, Cat# 74104) from cells and reverse transcription was performed. The resulting cDNA was diluted in nuclease free water. Finally, quantification was performed by real-time PCR using SYBR Green PCR mix (Eurogentec, Cat# RT-SY2X-03-WOU) and a Bio-RAD CFX Connect Real-Time PCR System. Silencing efficiency was assessed by comparing the gene of interest / Actin-B transcript ratio in control siRNA-treated cells versus silenced cells. The primer sequences used for quantitative real-time PCR were as follows: Murine LACC1 F 5’-TGGGGTTGCTCACTCCGGCTG-3’; R 5’-GGAGACTGCTGATTCTTTGGGAAGA-3’; Murine ActinB F 5’- GATGCTCCCCGGGCTGTATT-3’; R 5’-GGGGTACTTCAGGGTCAGGA-3'. For human cell lines, primers were used as described previously (1).

[0129] Mice

[0130] Age-, gender- and, whenever possible, littermate-matched 6- to 9-week old mice were used for all experiments, unless indicated otherwise. Mice were housed in a 19-21 °C environment and a 12 h light / dark cycle, and were fed a universal maintenance chow diet, purchased from Safe (Safe 105, SAFE® Complete Care Competence). Famin+ / +, Fami -, Faminp254', Faminp 254Vand Fam / np.284R mice and their wild-type littermate controls, which had all been generated on a C57BL / 6NTac background, have previously beendescribed (2, 3). IEC- (FaminmVil-Cre - ‘FaminMEC’) mice were generated by crossing Faminmmice with constitutively active Vil-Cre alleles.

[0131] Heterozygous breeding for the respective Cre alleles yielded their littermate controls. Each of these mouse lines and their controls were born at Mendelian ratio and developed normally under specific pathogen-free (SPF) conditions, which also applies to Famin mutant lines previously described (73). For spontaneous intestinal tumour development, ApcMmmice (C57BL / 6J-ApcMin / J mice, strain #002020, RRID:IMSR_JAX:002020, The Jackson Laboratory) were intercrossed with Fam / n^- mice. Maintenance and breeding under SPF conditions was performed at the Central Biomedical Services (CBS), Phenomics Laboratory, or the Anne McLaren Building at the University of Cambridge. Metabolic studies were performed at the Phenomics Laboratory animal facility. UK Home Office and local ethics approval has been granted for all experimental procedures.

[0132] Pancreatic islet isolation, culture and imaging

[0133] Mouse pancreatic islet isolations were performed by pancreatic ductal inflation with collagenase (Sigma Aldrich, Cat# C9263), followed by excision and digestion at 37 °C, mechanical disruption, and islet separation using a Ficoll-Paque plus gradient (Cytiva, Cat# 17144003). Islets were cultured in RPMI media (ThermoFisher, Cat# 31870) at 37°C (5% CO2).

[0134] Calcium imaging experiments were performed 2-3 days post islet isolation. Islets were loaded with 5 pM Fura-2 AM (Invitrogen, Cat# F1221) for 1 hour at 37°C, then washed 5 times prior to imaging to ensure washout of excess Fura. Islets were then imaged at room temperature using a 40* oil immersion objective (1.35NA) on an inverted microscope (Olympus 1X71 ) with Metafluor software (Molecular Devices) capturing images every 2 seconds during continuous perifusion of saline buffer solution in the presence of the indicated glucose concentrations with compounds or vehicle controls using a custom-made gravity-assisted perifusion apparatus with a flow rate of 2ml / minute. Saline buffer contained 138 mM NaCI, 4.5 mM KCI, 4.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.6 mM CaCI2, 1.2 mM MgCI2, 10 mM HEPES; adjusted to pH 7.4 with NaOH. Mean whole islet Fura-2 ratios (340 / 380 nm) were calculated following background subtraction in Metaflour software. Results were obtained from 5 independent islet cultures from 6 mice per genotype.

[0135] Western blot

[0136] For protein isolation in cell lines, cells were trypsinized (Trypsin-EDTA 0.05%, ThermoFisher, Cat# 25300054), washed once in PBS and resuspended in ice-cold RIPA buffer (50mM Tris pH8, 150mM NaCI, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) containing protease- and phosphatase-inhibitors. The lysate was incubated on ice for 30 minutes with gentle agitation, and spun down at 12,000xg for 10 minutes to remove cell debris. Protein content was measured by BCA (Pierce BCA Protein Assay Kit, ThermoFisher #23225). Equal amounts of protein eluted in Laemmli buffer (Bio-Rad, Cat# 1610747) were denatured at 95°C and resolved on 8-12.5% SDS-PAGE and transferred to a 0.2 pM Nitrocellulose membrane (Bio-Rad, #170-4159). After blocking in 5% skim milk, membranes were incubated with primary antibody (LACC1 Antibody (E-12), Santa Cruz Biotechnology, Cat# sc-376231 or beta Actin antibody (C4), Santa Cruz Biotechnology, Cat# sc-47778) in 5% BSA TBS-Tween at 4°C overnight. After washing 3x in TBS-Tween for 30 minutes each, membranes were incubated with secondary antibody (Anti-mouse IgG, HRP-linked Antibody, Cell Signaling Technology, Cat# 7076 or Goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed, Alexa FluorTM Plus 488, Invitrogen, Cat# A32731)for 1 hour at room temperature, and washed 3x again. The signal was visualized using 20X LumiGLO (Cell signaling, Cat# 7003).

[0137] Statistical analysis

[0138] Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software LLC, Version 9.4.0). Unless stated otherwise, statistical significance was calculated as appropriate using unpaired, two-tailed Student’s t-test, one-way analysis of variance (ANOVA). A P value of < 0.05 was considered to indicate statistical significance. Data are represented as individual data points with the mean and standard error of the mean (SEM). Schematic depictions were generated using BioRender.

[0139] Results

[0140] The purine nucleotide cycle (PNC) is an oscillating cycle between AMP, IMP, succinyl-AMP (S-AMP) and back to AMP, mediated by the cytoplasmic enzymes AMP deaminase (AMPD; AMP + H2O ->• IMP + NH3), adenylosuccinate synthase (ADSS; IMP + GTP + aspartate S-AMP + GDP + Pi) and adenylosuccinate lyase (ADSL; S-AMP AMP + fumarate) (9-72). The amino group re-aminating IMP to AMP stems from aspartate, whose carbons are released as fumarate (Fig 1a). Levels of S-AMP, which is not party to other reactions, were found to be 10-fold lower in the gene-edited FAMIN-deficient SW480FAMIN KOcell line, compared to its parent isogenic mock-gene edited colorectal cancer cell line SW480 (‘SW480WT’ for wildtype) (Fig 5a, Fig 1b).

[0141] In an attempt to measure flux through the PNC, we exploited efficient uptake of exogenous [13CT5N] aspartate by SW480 cells. A 3h pulse of 2 mM [13CT5N] aspartate (approximately equivalent to its reported intracellular concentration (73)) labelled -17-20% of cellular aspartate as [13CT5N] in both SW480WTand SW480FAMIN KOcells (Fig 1c). None of the [13C415N] aspartate incorporated into S-AMP (Fig 1d). This conclusion was limited by the low abundance of S-AMP, albeit we detected [13C4] S-AMP in some SW480WTsamples, i.e. formed from endogenously synthesised aspartate. Exogenous [13C415N] aspartate did not incorporate into argininosuccinate either, another cytoplasmic aspartate-consuming reaction (argininosuccinate synthase [ASS]: citrulline + aspartate + ATP argininosuccinate + AMP + PPi), which is part of the urea cycle (Fig 1a, 1e). Analogous to S-AMP, [13C4] argininosuccinate, i.e. incorporating [13C4] aspartate in the ASS reaction, however, represented 5% of total argininosuccinate in SW480WT cells, without any [13C4] incorporation detectable in SW480FAMIN KOcells. Total argininosuccinate levels in SW480FAMIN KOcells were modestly reduced compared to SW480WTcells (Fig 5b). This showed that cytoplasmic ADSS and ASS are, surprisingly, not accessible by exogenously provided aspartate. Aspartate entering the PNC and urea cycle required endogenous synthesis, implying an inaccessible ‘dark pool’ of 4-carbon units in the cytoplasm.

[0142] Indeed, after a 3h pulse of [13C415N] aspartate, -8% of aspartate was [13C4] and -4% [15N] labelled in SW480WTcells, i.e. having undergone at least two transamination reactions via oxaloacetate (Fig. 1c). Consistent with this, -6% of cellular glutamate was [15N] labelled (Fig. 1e). [13C4] and [15N] aspartate fractional incorporation was 20-fold reduced in SW480FAMIN KOcompared to FAMINWTcells, and [15N] glutamate collapsed to a similar extent. This suggested a non-redundant role of FAM IN in glutamate-oxaloacetate transamination via GOT1 and GOT2, part of the malate-aspartate shuttle (MAS) that imports e~ into mitochondria, and an anaplerotic route into the Krebs cycle (75, 76). [13C4] isotopomers of Krebs cyclemetabolites were ~20-fold lower in SW480FAMIN KOcompared to SW480WTcells (Fig. 1 g-k), their total levels unchanged (Fig. 5c-h). Whilst fractional [13C4] labelling of malate, fumarate, citrate and isocitrate was substantial (~6-10%) in wild-type SW480 cells, labelling of [13Ca] aKG and [13C2] succinate dropped off to only 1.2% and 0.8%, respectively, and was virtually absent in SW480FAMIN KOcells. This indicated that exogenous aspartate-derived 4-carbon units do not progress past isocitrate via the Krebs cycle (17). The mitochondrial membrane potential was markedly lower in SW480FAMIN KOcompared to SW480WTcells (Fig.

[0143] 11), consistent with FAMIN enabling flux of 4-carbon units into the MAS and hence e~ import into mitochondria. The MAS relies on SLC25A12 / SLC25A13-dependent exchange of aspartate for glutamate and a proton (H1; Fig. 1a) (15). Consistent with reduced MAS flux was cytoplasmic acidification (pHc) in SW480FAMIN KOcompared to SW480WTcells (Fig. 1m), a shared feature we had previously reported across a range of different FAMIN-deficient cell types (1, 3).

[0144] The MAS depends on transamination between 4-carbon (aspartate / oxaloacetate) and 5-carbon units (glutamate / aKG; Fig. 1a) (15). We therefore pulsed SW480 cells for 3 h with 2 mM [13C515N] glutamate, which labelled a third of cellular glutamate in SW480WTand SW480FAMIN KOcells (Fig. 2a). Consistent with FAMIN affecting transamination, [13Cs] glutamate, [13Cs] aKG and [15N] aspartate labelling halved in SW480FAMIN KOcompared to FAMINWTcells (Fig. 2a-c). [13C4] isotopomers of Krebs cycle intermediates and aspartate also halved in SW480FAMIN KOcompared to SW480WTcells (Fig. 2d). Altogether, this showed that FAMIN determined 5-carbon flux into the MAS and Krebs cycle. To help distinguish the latter from the former, we traced [13Cs15N2] glutamine, which is converted into glutamate via strictly mitochondrial glutaminase (GLS, glutamine ->• glutamate + NH3) and enters the Krebs cycle via glutamate dehydrogenase (GDH, glutamate + NAD1# aKG + NADH + NH3) or transamination via mitochondrial GOT2 (Fig. 1a) (18). A 3h pulse of [13CS15N2] glutamine labelled 98% of total glutamine in SW480WTand SW480FAMIN KOcells (Fig.

[0145] 6a), resulting in minimally increased (82 — 85%) fractional incorporation in [13Cs] aKG in SW480FAMIN KOcompared to SW480WTcells (Fig. 6b), and correspondingly slightly higher fractional labelling of the onward [13C4] isotopomers of Krebs cycle intermediates and aspartate (Fig. 6c-h). These data showed that 5-carbon units from glutamine and glutamate entered the MAS and Krebs cycle via distinct routes. S-AMP and argininosuccinate were almost entirely [13C4] labelled (Fig. 6i-j), demonstrating glutamine oxidation as the predominant source of aspartate entering the PNC and urea cycle. The results of the glutamine labelling also implied that anaplerosis into the Krebs cycle and flux through the Krebs cycle were largely unaffected by FAMIN. The latter was confirmed by tracing a 3 h pulse of [13C3] pyruvate (Fig. 6k-q) and [13C6] glucose (Fig.

[0146] 6r-x). Hence, FAMIN selectively controlled flux into and through the MAS via both its 4-carbon and 5-carbon pool while leaving Krebs cycle activity largely unaffected.

[0147] Mitochondrially synthesised aspartate can recirculate through the MAS via cytoplasmic GOT1 or supply the PNC and urea cycle (Fig. 1a). To test for metabolic channelling (19) between MAS, PNC and urea cycle, we tipped the GOT1 equilibrium toward reduced MAS flux to divert some synthesised aspartate to ADSS and ASS. Partial GOT1 inhibition (achieved by GOT1 -selective inhibitor GOT1-inh-2c [CAS No 732973-87-4] (20) added at its IC50 concentration for 3h) after a [13C415N] aspartate pulse increased [13C4] argininosuccinate and rendered [13C4] flux into S-AMP well-detectable (~3%) in SW480WTcells (Fig. 1d-e). None of these [13C4] isotopomers were detectable in SW480FAMIN KOcells. [13C415N] argininosuccinate and S-AMP remained undetectable in either genotype and [13C415N] aspartate uptake and transamination to [15N] glutamate were not affected by partial GOT1 inhibition (Fig. 1c, Fig .1 f). While total argininosuccinate remained unchanged onpartial GOT1 inhibition (Fig. 5b), it doubled levels of PNC metabolites S-AMP and IMP in SW480WTcells (Fig. 1b, Fig. 1n). This effect was dependent on FAMIN, since IMP levels remained unchanged and S-AMP markedly depressed, in SW480FAMIN KOcells. Importantly, total levels of hypoxanthine, IMP’S nucleobase and a FAMIN substrate were 40-fold higher, and inosine levels 3.5-fold lower, in SW480FAMIN KOcompared to SW480WTcells (Fig.1 0-p). These reciprocal nucleobase / nucleoside levels extended to FAMIN substrates guanine / guanosine (Fig. 1 q-r), suggesting FAMIN-dependent nucleoside synthesis from nucleobases as its prevailing catalytic direction. Partial GOT1 inhibition increased hypoxanthine and guanine levels in SW480FAMIN KO, and inosine and guanosine levels in SW480WTcells (Fig. 1 q-r). [13C4] isotopomers of Krebs cycle intermediates increased in GOT1 inhibitor-treated SW480WTcells after a [13C415N] aspartate pulse, whilst they remained severely depressed in SW480FAMIN KOcells (Fig. 1g-k). These results suggested that [13C4] fumarate released from PNC and urea cycle recirculated back into the MAS. It also showed how electron transport into mitochondria is closely intertwined with core purine turnover in a FAMIN-dependent manner. Altogether this suggested that the MAS, urea cycle and PNC operate as a unit, and purine nucleobase / nucleoside turnover via FAMIN is involved in enabling their flux.

[0148] We next asked how a complete blockade of GOT1 would affect the PNC and urea cycle. Applying GOT1-in-2c at its 10x IC50 concentration (100 pM) with a 3 h pulse of [13C415N] aspartate increased its fractional incorporation in both SW480WTand SW480FAMIN KOcells (Fig. 2h), compensating for lower endogenous synthesis reflected by lower total aspartate levels (Fig. 2i). Fractional labelling of [13C4] aspartate, i.e. having undergone exchange of its amine, increased upon GOT1 blockade in SW480WTcells, but remained severely depressed in SW480FAMIN KOcells (Fig. 2h). Total levels of S-AMP increased 7-fold upon GOT1 blockade, now independent of presence or absence of FAMIN (Fig. 2j), whilst levels of urea cycle-specific argininosuccinate dropped by 50% (Fig. 2k). GOT1 blockade in SW480WTcells increased [13C4] S-AMP labelling from undetectable to 12% (Fig. 2I) and [13C4] argininosuccinate from 2% to 13% (Fig. 2m). Neither of these isotopomers were detectable in SW480FAMIN KOcells. This marked boost in [13C4] fractional labelling upon complete GOT1 blockade in FAMIN-sufficient cells extended to fumarate and malate (Fig. 2n-o), i.e. the 4-carbon unit released from S-AMP and argininosuccinate. [13C4] citrate fractional incorporation was unaffected by GOT1 blockade in SW480WTcells (Fig. 2p), whilst [13C4] isocitrate (and [13C3] aKG and [13C2] succinate accordingly) labelling turned undetectable upon GOT1 blockade (Fig. 2q). As expected, total levels of all Krebs cycle metabolites were lower upon GOT1 blockade (Fig. 7 a-f). This showed that FAM IN was required to route much more exogenously provided aspartate-derived 4-carbons into the mitochondrial part of the MAS by shifting cytoplasmic flux, particularly to the PNC, when mitochondrial aspartate synthesis was impaired. This circulating 4-carbon MAS pool did not mix with the Krebs cycle.

[0149] The majority of 4-carbon units circulating via the MAS may be mitochondrially synthesised, i.e. derived initially from the Krebs cycle. To explore this, we pulsed SW480 cells for 3 h with [13Cs15N] glutamate in the presence and absence of 100 pM GOT1-inh-2c (Fig. 2a). Compared to SW480WTcells, [13C4] aspartate levels were similarly depressed in SW480FAMIN KOand GOT1 -blocked SW480WTcells, but completely absent in GOT1-blocked SW480FAMIN KOcells (Fig. 2c). Total aspartate levels declined accordingly (Fig. 2e). This pointed to an additive effect of GOT1 inhibition and FAMIN deficiency on glutamate carbon -derived aspartate synthesis. [13C4] isotopomers of Krebs cycle metabolites corroborated this pattern (Fig. 2d). Importantly, [13C4] isotopomers of S-AMP were exclusively detectable in GOT1-blocked SW480WTcells (Fig. 2f), whereas [13C4] isotopomers of argininosuccinate only in control-treated SW480WTcells but not GOT1 -inhibited cells(Fig. 2g). Hence, GOT1-dependent transamination to aKG routed glutamate-derived 4-carbons into the urea cycle, whereas its inhibition routed glutamate-derived 4-carbons into the PNC. In either case, FAMIN was non-redundant. Interestingly, GOT1 blockade almost completely rescued depressed [13Cs] aKG of SW480FAMIN KOcells (Fig. 2b), suggesting enhanced oxidative deamination of [13Cs15N] glutamate when transamination is blocked (Fig. 1a). However, anaplerosis via GDH did not, under normal GOT1 function, affect FAMIN-dependent routing of [13Cs15N] glutamate-derived stable isotopes as revealed by selective GDH inhibition (Fig. 8a-g), suggesting transamination as the predominant route into the MAS and Krebs cycle. Altogether, this showed that FAMIN was critical for feeding 5-carbon units into the Krebs cycle and MAS and that their oxidation yielded 4-carbon units entering the MAS, PNC and urea cycle.

[0150] The MAS transfers e~ onto complex I of the electron transport chain (ETC, Fig. 1a) (15, 21). Inhibiting complex I in SW480WTand SW480FAMIN KOcells pulsed for 3 h with [13C415N] aspartate (Fig. 2r-s) mirrored results obtained with GOT1 blockade: [13C4] S-AMP labelling in complex l-inhibitor treated SW480WTcells was boosted from undetectable to 21% (Fig. 2t), and [13C4] argininosuccinate increased 10-fold from 3% to 32% (Fig. 2u). Critically, [13C4] isotopomers of S-AMP and argininosuccinate remained undetectable in SW480FAMIN KOcells even after complex I inhibition, corroborating the non-redundant role of FAMIN in aspartate-derived 4-carbon flux through the PNC and urea cycle. The increase in total S-AMP levels upon complex 1 inhibition was dependent on FAMIN (Fig. 2v), whilst total argininosuccinate levels declined with FAMIN deletion and complex I inhibition (Fig. 2w). The marked boost in [13C4] isotopomers of fumarate and malate in complex I inhibitor-treated SW480WTcells and their complete absence in SW480FAMIN KOcells traced their origin to S-AMP and, secondarily, argininosuccinate cleavage (Fig.2 x-y). This showed that the collapse in mitochondrial aspartate synthesis due to complex I or GOT1 blockade pulled in more cytoplasmic aspartate carbon and re-routed 4-carbons primarily via the PNC. Hence, the MAS, PNC and urea cycle operated as one metabolic unit, and FAMIN was non-redundant for feeding 4-carbon and 5-carbon units into it.

[0151] The complete absence of [13C4] S-AMP, argininosuccinate and aspartate in FAMIN-deficient cells upon a [13C415N] aspartate pulse was startling. We therefore considered whether FAMIN’s already unusual catalytic multi-functionality extended to aspartate and oxaloacetate, specifically to transamination with aKG. Highly purified recombinant FAM IN (1) indeed exhibited robust aspartate-aKG (aspartate + aKG

[0152] oxaloacetate + glutamate) (Fig. 3a, Fig. 3b and reverse glutamate-oxaloacetate (glutamate + oxaloacetate aKG + aspartate) transaminase activity as demonstrated by LC-MS (Fig. 3b-c). Aspartate transaminase activity was absent with recombinant FAMIN-C284S, in which the active site cysteine is mutated to serine (Fig. 3d-e). Transaminase activity was also absent from heat-denatured FAMIN (Fig. 3f-g), and not present with an unrelated purine-metabolising recombinant enzyme, thiopurine methyltransferase (TPMT) (Fig. 3a, Fig. 3c). FAMIN specifically transaminated aspartate since no alanine transaminase (alanine + aKG pyruvate + glutamate) activity was detected (Fig. 3h). Transaminase activity was also present with FAMIN’s bacterial orthologue YlmD, but again abrogated by heat denaturation (Fig. 3i-j). Hence, aspartate transamination is evolutionarily conserved, akin to its purine nucleoside catalysis (1). Remarkably, while these new FAMIN activities are identical to those of cytoplasmic GOT1 and mitochondrial GOT2, ubiquitous enzymes much higher expressed in SW480 cells (22), FAM IN was completely non-redundant for enabling exogenous aspartate carbon flux via the MAS, PNC and urea cycle (Fig. 1 & 2). Whilst the selective GOT1 inhibitor GOT1-inh-2c left FAMIN’s aspartate transamination unaffected, the non-selective GOT inhibitoraminooxyacetate (AOA) potently inhibited it (Fig. 3k-l). Both this GOT-like activity of FAMIN, as well as its purine nucleoside deaminase and phosphorolytic cleavage activities are physiologically relevant, the latter evident by the up to 40-fold differences in purine nucleobase and -nucleoside levels in FAMIN-mutant cells (Fig. 1o-r).

[0153] FAMIN had been proposed to also cleave citrulline to ornithine and isocyanic acid (23), effectively suggesting a urea cycle short-cut (Fig. 1a). Citrulline and ornithine levels were indistinguishable between SW480WTand SW480FAMIN KOcells (Fig. 9a). Highly purified recombinant FAMIN and YlmD, including a maltose binding protein (MBP)-FAMIN fusion protein (7) that Wei et al had used (23), did not cleave citrulline into ornithine, while these proteins readily catalysed reactions on purine nucleosides (Fig. 9b-d Hence, we could not detect citrulline cleavage in our hands nor find evidence corresponding to such activity in FAMIN-mutant compared to wild-type cell lines.

[0154] A key, often overlooked feature of the PNC is its inherent oscillatory nature, most narrowly explained by the opposite energetic requirements of AMP deamination vs re-amination (9-72). Lowenstein and Tornheim demonstrated in particle-free muscle extracts 6-20 min coordinated oscillations in levels of IMP, S-AMP and AMP (Fig. 4a) (9, 24). These oscillations in PNC metabolites were in phase with oscillations in levels of glycolytic intermediates and NADH (9, 24-28) (Fig. 4a). As the primary control point, they inferred sudden transient bursts of phosphofructokinase (PFK-1) activity (72, 26, 28-31), the rate-limiting enzyme of glycolysis whose feed-forward allosteric regulation would be augmented and inhibited, respectively, by peaks in AMP and ATP levels (32) (Fig. 4a). Continuous monitoring of dynamic levels of multiple metabolites in intact, live cells or organs is impossible. However, there is one tissue / cell type in which bursts of PFK-1 activity, alongside oscillations in glycolytic metabolites, NADH, mitochondrial membrane potential and oxygen consumption occur synchronised across the organ. These are pancreatic p cell islets, where these metabolic oscillations are tied to the transient closing of plasma membrane ATP-dependent K+channels, oscillations in cytoplasmic calcium ([Ca2+]i), and consequent synchronised pulsatile insulin secretion (33-36). This is the basis of ~6 min interval oscillations in plasma insulin and glucose levels (37, 38). Hence, [Ca2+]i probes afforded an experimentally tractable system for testing whether these interlinked electro-metabolic oscillations of pancreatic islets are affected by FAMIN. All pancreatic islets from Faminp 254' (26 / 26; carrying a fully-active FAMIN allele (2)) and Fam / / ?P254V(13 / 13; carrying a partially-active allele) mice exhibited ‘slow’ [Ca2+]i oscillations upon increasing the glucose concentration from 2 to 11 mM in the perifusion medium (Fig.

[0155] 4b-c, 4e-f), the established method to elicit them in ex vivo culture. In contrast, only 5 of 25 islets (20%) from Faminp 284Rmice (carrying a catalytically-dead allele (2)) exhibited ‘slow’ [Ca2+]i oscillations under the same conditions (Fig. 4d, 4g). The addition of the ADSS inhibitor L-alanosine (Fig. 4b-d) or the AMPD inhibitor Cpd3 (Fig. 4e-g), which block the re-aminating and de-aminating legs of the PNC (7, 3), respectively, had an instantaneous impact on frequency in all islets in which 11 mM glucose had triggered ‘slow’ oscillations. In the majority of those, L-alanosine and Cpd3 caused a complete halt in oscillations, while in others the frequency of oscillations changed. This was in stark contrast to the 80% of islets from Fam / np.284R mice, in which 11 mM glucose had not triggered [Ca2+]i oscillations (Fig. 4d). In these, L-alanosine and Cpd3 had no apparent effect (Fig. 4d). Notably, in a few Faminp 254' islets in which L-alanosine had halted oscillations, stopping L-alanosine perifusion resulted in their instantaneous reverting to the original ‘slow’ oscillations (Fig.

[0156] 4H). This implied that a PNC is present in pancreatic islets, with FAMIN contributing to the probabilistic occurrence of oscillations.Aspartate is an endogenous metabolic limitation of tumour growth under hypoxic conditions ( 18, 40-43). Aspartate aminotransferases G0T1 and G0T2 can be redundant for tumour formation in select cancers since SLC1 A family transporter-mediated aspartate uptake or extracellular protein scavenging via macropinocytosis may bypass this limitation (44-47). Electron transfer to complex I plays a critical role in malignancy (48). FAMIN silencing halts the proliferation of the human hepatocellular carcinoma cell line HepG2 (1). Gene-deleted SW480FAMIN KOcells exhibited a substantial reduction in proliferation compared to SW480WTcells (Fig. 10a). Attempts to generate FAMIN-deficient lines of HepG2 cells or of the murine melanoma cell line B16F10 failed despite screening >300 individual clones seeded from high-efficiency bulktargeting, corroborating FAMIN’s non-redundant role. Transient silencing of the human breast cancer cell line MCF-7 or B16F10 cells resulted in profound proliferation defects (Fig. 10a). A 3 h pulse of [13C415N] aspartate in B16F10 cells 72 h after siRNA transfection tripled fractional incorporation in Famin- (29.4%) compared to mock-silenced (10.5%) B16F10 cells (Fig. 10b), leading to nominally similar, but relative to uptake, reduced incorporation of [13C4] units into the Krebs cycle in Famin- and control-silenced cells (Fig. 10c-e). Turning to 5-carbon anaplerosis, fractional incorporation in FAMIN- compared to control-silenced B16F10 cells aftera 90 min [13C515N] glutamate pulse increased [13C515N] glutamate (62% 83%) (Fig. 10f) and [13Cs] aKG (68% — 82%) fractional labelling (Fig. 10g), whilst resulting in equal levels of onward-oxidised [13C4] succinate (Fig. 10h). However, [13C4] aspartate labelling collapsed from 12% to 1% (Fig. 10i), [13C4] citrate from 10% to 0.6% (Fig. 10j) and [13C4] isocitrate from 3% to 0% (Fig. 10k) in FAMIN- compared to control-silenced B16F10 melanoma cells, with corresponding 3-fold reductions in [13C4] fumarate and malate (Fig.

[0157] 101-m). S-AMP levels were too low to assess [13C4] incorporation, but [13C4] argininosuccinate collapsed in FAMIN- compared to control-silenced B16F10 cells (Fig. 10n), corroborating the FAMIN-dependency of 4-carbon flux through an integrated MAS / PNC / urea cycle. FAMIN- compared to control-silenced cell lines again exhibited a reduction in mitochondrial membrane potential (Fig. 10o) and cytoplasmic acidification (Fig.

[0158] 10p), consistent with the MAS’ direct and indirect role in driving mitochondrial H+import (15). Aspartate, glutamate, glutamine, or pyruvate did not rescue the proliferation defect in SW480FAMIN KOcells (Fig. 10q), nor did the supply of purine nucleosides or -nucleobases (Fig. 10r-s). Altogether this confirmed a FAMIN-enabled dynamic process rather than a static deficiency of catalytic product(s) caused by its absence.References

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[0240] 1 maeavlidlf glklnsqknc hqtllktlna vqyhhaakak flcimccsni syerdgeqdn 61 ceietsngls alleefeivs cpsmaatlyt ikqkideknl ssikvivprh rktlmkafid 121 qlftdvynfe fedlqvtfrg glfkqsiein vitaqelrgi qneietflrs Ipalrgklti 181 itsslipdif ihgfttrtgg isyiptlssf nlfssskrrd pkvvvqenlr rlanaagfnv 241 ekfyrikthh sndiwimgrk epdsydgitt nqrgvtiaal gadcipivfa dpvkkacgva 301 hagwkgtllg vamatvnami aeygcsledi vvvlgpsvgp ccftlpresa eafhnlhpac 361 vqlfdspnpc idirkatril leqggilpqn iqdqnqdlnl ctschpdkff shvrdglnfg 421 tqigfisike

[0241] SEQ ID NO: 1 Homo sapiens FAMIN amino acid sequence (NP_001121775.1)

[0242] 1 agacctgcag ctcctgccgc cctgcgcccg ctcccagggc ccgtcgttcc gccgccctat 61 ccctcctcaa ggggccccta gctgcctcct cgcgaccctt tccggactcg gcctgcccac 121 tcctgcccgc taacccgcct ggctcccggg cgagagccct cgcgcggctc tggttcctgt 181 tcctctaacg ccgccggggc tgcgggatgc cgactccgcg gaccgcccag acccggaact 241 gctgaggcag cagcgggctc gcggcgcttg gctcatcccg ggattcccca gctctcgcgc 301 tgggcccgcc gcgttcgcac caagcacgcc aggcggccct ggcctacctc cctcccgcct 361 cccggcagct ggcacgaggg aacctggccg tcaggtttcc cctgggatcc tgggacggta 421 tcaggcgggg aatctgtgcg gccgcggcga ggtgatttat ttggcataaa agtattcttt 481 caaggatggc agaagctgtt ttgattgatc tttttggttt gaaattgaac tctcaaaaaa 541 actgccatca gacattactg aagactttga atgctgtcca ataccaccat gctgccaagg 601 ccaagtttct ctgtataatg tgttgcagta acatcagcta tgaaagggat ggagaacaag 661 ataattgtga aatagaaaca agcaatggat tatcagctct cttggaagaa tttgagattg 721 ttagctgtcc cagcatggct gccactttgt ataccattaa acagaaaatt gatgaaaaaa 781 atctgagcag cattaaggta attgtaccca ggcacaggaa gacattaatg aaagctttta 841 ttgatcaact cttcactgat gtttacaatt ttgaatttga agatttgcaa gtgactttta 901 ggggagggct ttttaaacag tccattgaaa taaacgtaat cacagctcaa gaactaagag 961 gaattcagaa tgaaatagaa acatttttga gaagtctgcc agcactgaga ggaaaattaa 1021 ctattatcac ttcttctttg atcccagata ttttcataca tggatttact acaagaacag 1081 gtgggatatc ttatatacca actcttagct cattcaatct cttcagtagt tccaaacgga 1141 gagatcccaa ggtagtggtt caagaaaatc tgcgtaggtt ggcgaatgct gcaggattta 1201 atgtggagaa attttaccga ataaagactc atcattccaa tgacatctgg attatgggaa 1261 gaaaggagcc tgactcttat gatggaataa ccacaaatca gagaggagtc acaatagcag 1321 ctcttggtgc agactgtata ccgatagttt ttgcagatcc agtcaaaaaa gcatgtgggg 1381 ttgctcacgc tggttggaaa ggtactttgt tgggtgttgc tatggctaca gtgaatgcta 1441 tgatagcaga atatggctgc agtttggaag acattgttgt tgtacttgga ccttcagtag 1501 gaccttgctg ttttactctt ccaagggaat cagcagaggc atttcataat cttcatcctg 1561 catgtgtaca actatttgat tcaccaaatc cctgtatcga catccgtaaa gccacaagga 1621 ttcttctaga acagggagga attcttccac agaatattca ggaccagaac caagatctca 1681 acctctgtac atcttgccat cctgacaagt ttttctccca tgtccgagat ggccttaatt 1741 ttggtacaca gattggcttc atatcaatta aagaatgaga tacttgactg gatttttgta 1801 taactgcttc ctgcctcctt ccaaactgac tgcaagagag aaatttagct gtttgattta 1861 cttaaaacca aatggattac aatggataat tcatcttttg ggtatatttt tactattatt 1921 caaagccaaa tgattttcat ttaattgtaa taataactga caaaaatcag tatgttgtag1981 ctaatatgtt ttatgcatga gaattattct taaagtttgt tctccctgtt tattacacag 2041 atcaggaata gatttgttca gttcagtatt tattggatac cctctattgg tcaggcattg 2101 tgttaagcat atgtgaatca aaatgaacac aactttttcc tttgagtctg atacagtgaa 2161 ggagataaac acttctacaa cttaaattta attttaatag cagtagaaga gaacataagg 2221 aatagaggtt aattttaccc agaagcagga tagagaaaat attacagaga aaatcacata 2281 tcacatgggc tcgaaagatg tagaggtttt tgacaaatga agaacaacca taacaggtag 2341 agggaacacc atgaaccagg gcatgaaact gaaagtgcat aacatattct agagagagaa 2401 gggtgtgggc atgagttagg gctggaaaaa caggttggaa acagataagt aagggtctca 2461 aatgcaatgt caaagagctt gcagtttatt ttccaggcaa tgagtaggca gccaaaaaaa 2521 aaaaagtaag gatgtttttt ttttttttcc catggcatca tatttaagag gatggattta 2581 aattgtgtga gaccaaagca tagagactag ataagaggcg atcaaaatat ttcaaaaaga 2641 aataatgaag atccaatgaa ggaagtggaa attaaaatag ggaagagagt agatggatta 2701 gagagacatt taagagatgg aatcaataga tcctgttact agataatgga agtaagaggt 2761 gaggaagagt ggaaaagtca ttaatgactc taagatttct gcttggctgc ttaccaagat 2821 tggcaacaaa gggagggaga aggtttggaa aaagagagaa ggataatgag tttgacttta 2881 catagaatga agggcatcca gatagaaatc tttggttaat aattagaaat atagacctag 2941 aaattaggag gaaacctgag acagagacaa atatttcaaa gcttacaata cagagatgat 3001 acctgattct attggagcag gtttgatcat ctaggcagaa attaggatga gaaaaaagga 3061 gatccaataa tacaacctta tagtcacaga agtaagaaaa aaagggtagt tgttttgaag 3121 aagccaggat aggtgtggaa agtactcaaa aagaaatctt cagggataaa ataaagtgat 3181 aatttaaaag aaatcaatgg attaaacata ttgaaactgt tctataggca gtggtcattg 3241 agtcagcttt cagtgcatta ggaagaagat gcataggtgt caactctttt ctgacagcat 3301 ttactagaga agagaaaaag ctggggacta catcttcaag gaagggactt tttttggatg 3361 agcagttttg agtgtgtttg tcagttaaag agaggaatta ggttagtttt catttgggaa 3421 aaattgtata tatatttaat gtaagttatc acattgcatc ttaaaaatat tcttatttaa 3481 tacatatatt tcctacatgt atatgtggta gcatgatagc aaataacatt tgtttggtat 3541 ttccaaagga ctttcatgta cattgcctca ttttaccttt acagctactc tgaaatacac 3601 aggcattatc ccttttattc agctgagaaa actgagcttc attgaggtgg aggtcaaaaa 3661 tcacaaaatt tgtgatgaat taagatttga acatatgttt tgtgactcca gttttccttt 3721 cagattttaa aattaattaa agggatcttc attatacttt tattgttaac tttttgttaa 3781 cataatttat tcatacattc agtgaaaatt ttgttgaggt actgggacag gttaaaaaat 3841 acagttgtag ccctcaggat atttaatatc cagtgaaaag tgacagtcag taaaccaaca 3901 atctcaatac tttgatatat gttgtgaggt tgtgataacc gattcttgtt tagtttaatt 3961 ctatatctcc cttagaccag tgttaaattt aaataaaaca cctcattttt tccaattcag 4021 ggaaggcact aaacataaag cataggatag aaatgttgaa ctcatccaaa atattatttt 4081 gtttaatgaa aatgatgaag attaaggaat acttccatgt attgagtaag gttgataatt 4141 ttctaattct tcactgtgca ttattttgtt tgaagttggt aaatttggag tatcctgcag 4201 acacattttg ctttatgtac tacaacattc tacaaccaaa taaaaattat tttgattatc 4261 tgaaaaaaaa aaaaaaaaaa aaaaaaaa

[0243] SEQ ID NO: 2 Homo sapiens FAMIN coding sequence (NM_001128303.1)

Claims

35Claims1. A method of measuring the activity of a FAMIN protein comprising;providing a FAMIN protein; anddetermining the aspartate transaminase activity of the FAMIN protein.

2. A method of screening fora compound that modulates the activity of a FAMIN protein comprising;determining the aspartate transaminase activity of a FAMIN protein in the presence and absence of a test compound,wherein a difference in the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of test compound is indicative that the test compound modulates the activity of the FAMIN protein.

3. A method according to claim 2 wherein a decrease in the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of the test compound is indicative that the test compound is a FAM IN inhibitor.

4. A method according to claim 2 wherein an increase in the aspartate transaminase activity of the FAMIN protein in the presence relative to the absence of the test compound is indicative that the test compound is a FAM IN potentiator.

5. A method according to any one of the preceding claims wherein the FAM IN protein is an isolated FAMIN protein.

6. A method according to claim 5 wherein the method comprises contacting the FAM IN protein with aspartate and a-ketoglutarate.

7. A method according to claim 6 wherein the aspartate and / or a-ketoglutarate are labelled, optionally isotopically labelled.

8. A method according to claim 6 or claim 7 wherein the method comprises measuring the consumption of aspartate and / or a-ketoglutarate.

9. A method according to any one of claims 6 to 8 wherein the method comprises measuring the production of oxaloacetate and / or glutamate.

10. A method according to claim 9 wherein the oxaloacetate and / or glutamate are labelled, optionally isotopically labelled.

11. A method according to claim 10 wherein the aspartate is [13C415N] aspartate, the oxaloacetate is [13C4] oxaloacetate, and / or the glutamate is [15N] glutamate.3612. A method according to claim 5 wherein the method comprises contacting the FAMIN protein with oxaloacetate and glutamate.

13. A method according to claim 12 wherein the oxaloacetate, and / or glutamate are labelled, optionally isotopically labelled.

14. A method according to claim 12 or 13 wherein the method comprises measuring the consumption of oxaloacetate and / or glutamate.

15. A method according to any one of claims 12 to 14 wherein the method comprises measuring the production of aspartate and / or a- ketoglutarate.

16. A method according to claim 15 wherein the aspartate and / or a-ketoglutarate are labelled, optionally isotopically labelled.

17. A method according to claim 16 wherein the glutamate is [13C415N] glutamate, the aspartate is [15N] aspartate, and / or the a-ketoglutarate is [13Cs] a-ketoglutarate18. A method according to any one of claims 8 to 11 or claims 14 to 17 wherein consumption and / or production is measured using NMR, mass spectrometry, spectrophotometry, chemical assay methods or enzymatic assay methods.

19. A method according to claim 18 wherein consumption or production is measured using LC-MS.

20. A method according to any one of claims 5 to 19 wherein the isolated FAMIN protein is purified.

21. A method according to any one of claims 5 to 20 wherein the isolated FAMIN protein is recombinant.

22. A method according to any one of claims 5 to 21 wherein the FAMIN protein comprises an amino acid sequence having at least 80% identity to the sequence of amino acids 176-430 of NP_001121775.1 (SEQ ID NO. 1).

23. A method according to any one of claims 1 to 4 wherein the FAMIN protein is within a cell.

24. A method according to claim 23 wherein the cell is FAMIN proficient.

25. A method according to claim 23 or claim 24 wherein aspartate transaminase activity is determined by contacting the cell with one or more labelled substrates of the forward or reverse aspartate transaminase reaction and measuring the amount of one or more labelled metabolites of the substrates in the cell.

26. A method according to claim 25 wherein the labelled substrate is isotopically labelled aspartate.

27. A method according to claim 26 wherein the isotopically labelled aspartate is [13C415N] aspartate.

28. A method according to claim 26 or 27 wherein the labelled aspartate metabolites are isotopically labelled forms of aspartate, S-AMP, argininosuccinate, fumarate, malate, citrate, isocitrate, a-ketoglutarate, or succinate29. A method according to claim 28 wherein the isotopically labelled aspartate metabolites are one or more of [13C415N] aspartate, [13C4] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, [13C4] citrate, [13C4] isocitrate, [13Ca] a-ketoglutarate, or [13C2] succinate.

30. A method according to claim 29 wherein the isotopically labelled aspartate metabolites are one or more of [13C4] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, or [13C4] citrate.

31. A method according to claim 25 wherein the labelled substrate is isotopically labelled glutamate.

32. A method according to claim 31 wherein the isotopically labelled glutamate is [13Cs15N] glutamate.

33. A method according to claim 31 or 32 wherein the isotopically labelled glutamate metabolites are isotopically labelled forms of glutamate, a-ketoglutarate, aspartate, S-AMP, argininosuccinate, fumarate, malate, citrate, isocitrate, or succinate.

34. A method according to claim 33 wherein the isotopically labelled glutamate metabolites are [13Cs] glutamate, [13Cs15N] glutamate, [13Cs] a-ketoglutarate, [15N] aspartate, [13C4] aspartate, [13C415N] aspartate, [13C4] S-AMP, [13C4] argininosuccinate, [13C4] fumarate, [13C4] malate, [13C4] citrate, [13C4] isocitrate, or [13C4] succinate.

35. A method according to any one of claims 25 to 34 wherein the amount of isotopically labelled metabolites is measured using LC-MS.

36. A method according to any one of claims 23 to 35, wherein the cells are mammalian cells.

37. A method according to claim 36 wherein the cells are human cells.

38. A method according to claim 37 wherein the cells are human colorectal cancer cells.

39. A method according to claim 38 wherein the cells are human SW480 cells.

40. A method according to any one of claims 23 to 39 wherein the FAMIN protein is human FAMIN41. A method according to any one of claims 23 to 40 wherein the FAMIN protein comprises an amino acid sequence having at least 80% identity to the sequence of amino acids 176-430 of NP_001121775.1 (SEQ ID NO. 1).

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