Probe for researching working mechanism of NAD (Nicotinamide Adenine Dinucleotide) or metabolite thereof and application thereof
By combining azide-modified nicotinamide adenine dinucleotide with a biotin EBT probe, and utilizing photocrosslinking and enzymatic reactions, the accuracy problem caused by the binding differences of existing probes was solved, enabling more accurate research on the working mechanism of NAD or its metabolites.
Patent Information
- Application Number
- CN202410926374.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-11
- Publication Date
- 2026-01-13
AI Technical Summary
In existing studies on the working mechanism of NAD or its metabolite cADPR, the differences in probe binding to proteins lead to insufficient accuracy of the results, making it difficult to accurately reflect the true interacting proteome.
Azide-modified nicotinamide adenine dinucleotide or its metabolites were combined with biotinylated EBT probes to achieve specific binding of interacting proteins via photocrosslinking and ADP ribosylcyclase catalysis. Streptavidin was then used for immunoprecipitation and proteomic identification.
This improves the accuracy and reliability of studies on the working mechanism of NAD or its metabolites, enabling more accurate capture of interacting proteins and reflecting their functions and roles in cells.
Smart Images
Figure CN121324656A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of nicotinamide adenine dinucleotide and its metabolites detection and research technology, and in particular to a probe for studying the working mechanism of NAD or its metabolites and its application. Background Technology
[0002] The role of SARM1 in axonal degeneration remains to be investigated. Its activation generates multiple signaling molecules, including cADPR and ADPR, initiating a SARM1-dependent cell death mechanism (sarmoptosis) with an unknown mechanism. This process is associated with calcium signaling release and mitochondrial dysfunction. Knocking out SARM1 inhibits NAD (nicotinamide adenine dinucleotide) depletion and calcium signaling pathways, thereby delaying axonal degeneration. cADPR, as a metabolic molecule catalyzed by SARM1 and an important calcium messenger, is currently considered an important indicator of SARM1 activation, but its role in axonal degeneration remains unclear. Furthermore, cADPR is associated with functions such as insulin secretion, cellular immunity, and muscle contraction, but the specific mechanisms also require further investigation.
[0003] NAD(P) is an important electron carrier in redox reactions, responsible for many vital cellular functions, including biosynthesis, energy metabolism, DNA repair, and cell division. Impaired NAD(P) synthesis and metabolism can lead to serious diseases, including cancer and neurological disorders. Furthermore, aging is accompanied by a decrease in NAD. The functions of NAD(P) are highly diverse, and many remain unknown and require further investigation. Both cADPR and NAD(P) rely on the regulation of protein activity and function for their proper function. Therefore, identifying the interacting proteins of cADPR and NAD(P) and elucidating their mechanisms of action in cellular physiological and pathological processes is an important approach and method for studying the working mechanisms of NAD or its metabolite cADPR.
[0004] Currently, methods for identifying unknown molecular targets mainly include: target-based chemical probe binding proteometry, phenotype-based CRISPR / Cas9 genome-wide screening, and bioinformatics analysis based on known protein and target binding data. In the absence of suitable phenotypic characteristics and known data, chemical probe binding proteometry is a suitable choice for identifying unknown cADPR and NAD interacting proteins.
[0005] Justina In their 2021 article, "Chemical Proteomics Approach for Profiling the NAD Interactome," they proposed using photoaffinity probes 2-ad-BAD or 6-ad-BAD to study the working mechanism of NAD. The structural formulas of 2-ad-BAD and 6-ad-BAD are as follows:
[0006]
[0007] In their 2017 article, "Identifying Glyceraldehyde 3-Phosphate Dehydrogenase as a Cyclic Adenosine Diphosphoribose Binding Protein by Photoaffinity Protein-Ligand Labeling Approach," Kehui Zhang et al. used the photoaffinity probe PAL-cIDPRE to study the working mechanism of cADPR. The structural formula of PAL-cIDPRE is as follows:
[0008]
[0009] However, the 2-ad-BAD and 6-ad-BAD probes have significant structural differences from NAD, as do the PAL-cIDPRE probe and cADPR. The proteins captured by these probes are affected by these structural differences, resulting in differences between the proteins bound by these two probes and the proteins that actually interact with NAD or cADPR. This, in turn, affects the accuracy of research results on the working mechanism of NAD or its metabolite cADPR.
[0010] Therefore, developing better and more accurate probes to more effectively study the working mechanisms of NAD or its metabolite cADPR remains a key research focus and challenge in this field. Summary of the Invention
[0011] The purpose of this application is to provide a novel probe for studying the working mechanism of NAD or its metabolites and its applications.
[0012] The following technical solution is adopted in this application:
[0013] One aspect of this application discloses a probe for studying the working mechanism of NAD or its metabolites. The probe is a dual-probe combination, comprising a first probe and a second probe. The first probe is an azide-modified nicotinamide adenine dinucleotide or an azide-modified nicotinamide adenine dinucleotide metabolite, wherein an azide group is introduced at the 8-position of adenine in the nicotinamide adenine dinucleotide or the nicotinamide adenine dinucleotide metabolite, so that the azide group forms a photocrosslinking reactive group with the adenine of the nicotinamide adenine dinucleotide or the nicotinamide adenine dinucleotide metabolite itself. The second probe is a biotin-containing EBT probe, the structural formula of which is shown in Formula 1.
[0014] It should be noted that this application is the first to creatively combine azide-modified nicotinamide adenine dinucleotide (NAD) or its metabolite with a biotin-based EBT probe, utilizing the specific binding and interacting proteome of the dual-probe system to study the working mechanism of NAD or its metabolites. The first probe of this application, namely the azide-modified NAD or its metabolite, has a structure closer to that of NAD or its metabolites; that is, it simply introduces an azide group at the 8-position of its adenine group. Therefore, the proteins hooked by this probe are more likely to actually interact with NAD or its metabolites. Thus, the probe of this application can more accurately and realistically reflect the working mechanism of NAD or its metabolites.
[0015] In one implementation of this application, the azido-modified nicotinamide adenine dinucleotide metabolite is azido-modified NADP, azido-modified cADPR, or azido-modified NAADP.
[0016] In one implementation of this application, the NADP modified with azide is shown in Equation 2.
[0017] In one implementation of this application, the azide-modified cADPR is shown in Equation 3.
[0018] In one implementation of this application, the azide-modified NAADP is as shown in Equation 4.
[0019] In one implementation of this application, the azide-modified nicotinamide adenine dinucleotide is shown in Formula 5.
[0020] Another aspect of this application discloses the application of the probe of this application in the detection of interacting proteins of NAD or its metabolites, or in the study of the working mechanism of NAD or its metabolites.
[0021] Another aspect of this application discloses a method for studying the working mechanism of NAD or its metabolites using the probes described in this application.
[0022] In one implementation of this application, the working mechanism research method includes firstly covalently crosslinking the first probe with the interacting protein through photocrosslinking reactive groups; then, using ADP ribose cyclase to exchange and link the second probe to the first probe; and finally, using streptavidin to perform immunoprecipitation, Western blotting, or proteomic identification of the interacting protein.
[0023] In one implementation of this application, the first probe is covalently cross-linked with the interacting protein by photocrosslinking reactive groups, specifically including, under ultraviolet light catalysis, covalently cross-linking the photocrosslinking reactive groups of the first probe with the interacting protein.
[0024] Another aspect of this application discloses a method for detecting interacting proteins of NAD or its metabolites using the probes described in this application.
[0025] It is understood that the method for detecting NAD or its metabolites interacting proteins using the probes of this application actually involves covalently cross-linking the first probe with the interacting protein, then exchanging the second probe with the first probe, and finally using streptavidin to perform immunoprecipitation, Western blotting, or proteomic analysis on the interacting protein. This can be understood by referring to the working mechanism research method of this application.
[0026] The beneficial effects of this application are as follows:
[0027] This application uses azide-modified nicotinamide adenine dinucleotide or its metabolite as a first probe to capture the interacting proteome, and uses biotin-labeled EBT as a second probe to label the first probe with biotin. Finally, biotin is used for the detection of the interacting proteome. The first probe in this application has a molecular structure closer to NAD or its metabolites; therefore, the captured interacting proteins more accurately reflect the actual binding of NAD to the interacting proteome, thus providing a more accurate and realistic representation of the working mechanism of NAD or its metabolites. Attached Figure Description
[0028] Figure 1 This is a flowchart illustrating the identification of interacting proteins using a dual-probe combination in an embodiment of this application;
[0029] Figure 2 This is the detection result of 8N3-cADPR-tagged CD38(E226Q) in the embodiments of this application;
[0030] Figure 3 This is the detection result of 8N3-cADPR-labeled recombinant protein cyclase in the embodiments of this application;
[0031] Figure 4 This is the detection result of 8N3-NADP-labeled CD38(E226Q) in the embodiments of this application;
[0032] Figure 5 This is the workflow for identifying interacting proteins using EPL-MS in the embodiments of this application;
[0033] Figure 6 These are the Western blot results of 8N3-cADPR-labeled cytoplasmic proteins of Ins-1E and HEK293 cell lines in the embodiments of this application;
[0034] Figure 7 This is the Western blot result of total protein from the 8N3-cADPR-labeled Ins-1E cell line in the embodiments of this application;
[0035] Figure 8 The results of mass spectrometry analysis of SARM1 and GAPDH in 8N3-cADPR-labeled Ins-1E in the embodiments of this application are shown.
[0036] Figure 9 This is the Western blot result of 8N3-cADPR, immunoprecipitated dN-SARM1, and OVA labeled in the embodiments of this application;
[0037] Figure 10 This is the Western blot result of 8N3-NADP-labeled HEK293 cell cytoplasmic proteins in the embodiments of this application;
[0038] Figure 11 This is the clustering result of 8N3-NAD and 8N3-NADP specific marker proteins in the embodiments of this application. Detailed Implementation
[0039] NAD (nicotinamide adenine dinucleotide) is an important coenzyme and substrate molecule in organisms, participating in important pathways such as energy metabolism, DNA repair, and cellular homeostasis. To further investigate the working mechanism of NAD, we have developed a highly efficient and specific NAD interactome identification technique, which can be widely used to identify the interacting proteome of NAD and its metabolites (NADP, cADPR, and NAADP).
[0040] Taking NAD as an example, this scheme transforms NAD into a photoaffinity-labeled probe through chemical modification, followed by UV cross-linking to covalently cross-link with the protein. To maintain the unique biological activity of NAD, the photoaffinity probe should minimally alter the original molecule's structure. Therefore, this scheme introduces an azide group at the 8-position of adenine in NAD, making the azide group and NAD's own adenine a photocrosslinking reactive group. The probe design approach for NAD metabolites NADP, cADPR, and NAADP is the same. Finally, our designed azide-modified nicotinamide adenine dinucleotide is shown in Equation 5, azide-modified NADP in Equation 2, azide-modified cADPR in Equation 3, and azide-modified NAADP in Equation 4.
[0041] Formula 2 Formula 3
[0042] Formula 4 Formula 5
[0043] Next, we developed a specific nucleophilic reagent composed of a nucleophilic group and biotin, named the Exchangeable Biotin Tag (EBT), i.e., an EBT probe carrying biotin. Utilizing the base substitution activity of ADP-ribosyl cyclase, the EBT can catalyze a molecular reaction between NAD or its metabolites, thus specifically attaching the affinity tag to NAD or its metabolites. The structural formula of the EBT probe is shown in Formula 1.
[0044] Formula 1
[0045] Therefore, it is ultimately applied to protein-protein interaction identification protocols such as Figure 1 As shown, firstly, under ultraviolet light catalysis, NAD or its metabolites are covalently cross-linked with interacting proteins via photocrosslinking reactive groups. Next, the EBT probe is attached to the protein using ADP-ribose cyclase. Finally, streptavidin is used for immunoprecipitation, Western blotting, and proteomic identification of the interacting proteins.
[0046] Furthermore, recent research has found that NAD, acting as a 5' cap on RNA, may participate in regulating RNA metabolism and function. Therefore, this technique can be applied to the identification of NAD-RNA, promoting research into its working mechanism. Similarly, for NAD-RNA identification, we used the developed exchangeable biotin tag (EBT) and cyclase base substitution reaction to introduce an affinity tag into NAD-RNA. After affinity purification using streptavidin, the NAD-RNA was identified using high-throughput RNA sequencing. In addition, CD38 pretreatment was used to eliminate interference from abundant intracellular m7G-RNA, further enhancing the method's specificity. This method is also applicable to the identification of other NAD analogue-modified RNAs.
[0047] The present application will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.
[0048] Example
[0049] I. Establishing a labeling method using the known interactions between cADPR and CD38(E226Q)
[0050] In this experiment, NAD was chemically modified into a photoaffinity-labeled probe, which was then covalently cross-linked with proteins after UV cross-linking. To maintain the unique biological activity of NAD, the photoaffinity probe should minimize changes to the original molecule's structure. Therefore, this scheme introduced an azide group at the 8-position of adenine in NAD, making the azide group and NAD's own adenine a photocrosslinking reactive group. The probe design for the NAD metabolites NADP, cADPR, and NAADP followed the same approach. Finally, the azide-modified nicotinamide adenine dinucleotide designed in this experiment is shown in Formula 5 (labeled as 8N3-NAD), the azide-modified NADP is shown in Formula 2 (labeled as 8N3-NADP), the azide-modified cADPR is shown in Formula 3 (labeled as 8N3-cADPR), and the azide-modified NAADP is shown in Formula 4 (labeled as 8N3-NAADP).
[0051] Formula 2 Formula 3
[0052] Formula 4 Formula 5
[0053] This experiment established a specific labeling method by designing and synthesizing an exchangeable biotin tag (EBT) and incorporating an ADP-ribose cyclase (E98G) enzymatic exchange reaction. The experimental procedure is as follows: Figure 1As shown, the protein was first incubated with the photocrosslinkable labeling probe (probe 1) to undergo a photocrosslinking reaction. After removing excess probe, cyclae (E98G) catalyzed an exchange reaction between EBT and the probe, linking biotin to the probe via a one-step enzymatic reaction. The results are as follows. Figure 2 As shown, the labeling efficiency of CD38(E266Q) after the addition of probe 1 was significantly higher than that of the probe-free group; although the amount of BSA protein in the system was much higher than that of CD38(E226Q), BSA was hardly labeled, indicating that the labeling of CD38 is photoaffinity probe-dependent and has very high specificity.
[0054] The structural formula of the EBT probe is shown in Formula 1.
[0055] Formula 1
[0056] Figure 1 and Figure 2 This demonstrates the two-step labeling method for 8N3-cADPR. Figure 1 The procedure is a two-step labeling method. Photoaffinity labeling covalently crosslinks the probe to the target protein, and biotin is introduced by the reaction of EBT and probe 1 catalyzed by cyclase (E98G). Figure 2 The results are from a Western blot analysis of 10 μM 8N3-cADPR, 1 μg CD38 (E226Q), and 15 μg BSA (EBT: 200 μM). Biotin signal was detected by Strep-HRP. The experiment was repeated three times. Statistical analysis was performed using unpaired t-tests. All calculated values are mean ± standard deviation. Statistical significance was defined as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).
[0057] To further verify the specificity of this labeling method, the labeling efficiency against another known cADPR-binding protein cyclase was examined, and a competition group treated with the original ligand cADPR was added to inhibit probe binding. The results are as follows: Figure 3 As shown, Figure 3 The results showed that the addition of the probe resulted in very high cyclase labeling efficiency, the addition of cADPR weakened the labeling signal, and no labeling signal was detected in the probe-free group. Ovalbumin (OVA) served as a negative control, and only a very weak non-specific signal was detected. These results indicate that cyclases can be specifically labeled by this 8N3-cADPR labeling method. In summary, the known cADPR recombinant protein labeling results demonstrate that this labeling method has high efficiency and specificity, and can be applied to subsequent studies of interacting proteins.
[0058] Figure 3 The labeling results of the 8N3-cADPR probe on the recombinant protein cyclase are shown. Specifically, the Western blot results are obtained after two steps of labeling with 5 μM 8N3-cADPR, 1 μg cyclase, and 15 μg OVA; cADPR: 250 μM, EBT: 200 μM.
[0059] Subsequently, it was verified whether this labeling method was also applicable to the labeling of 8N3-NADP-binding proteins. The results are as follows: Figure 4 As shown, the results for 8N3-NAD and 8N3-NADP are basically consistent with those for 8N3-cADPR. With the addition of the probe, CD38(E226Q) was clearly labeled. The addition of substrates NADP and NAD significantly inhibited CD38(E226Q) labeling, indicating that the labeling of CD38(E226Q) by 8N3-NAD and 8N3-NADP is specific. The amount of BSA protein was 20 times that of CD38(E226Q), but no labeling signal was detected. No labeling signal of CD38(E226Q) was detected in the probe-free group either, indicating that the non-specific labeling by 8N3-NAD and 8N3-NADP methods is minimal. Although CD38(E226Q) can bind to both NADP and NAD, the inhibitory effect of adding NADP on labeling is higher than that of NAD, indicating that the binding affinity of CD38(E226Q) to NADP is higher than that to NAD. These results demonstrate that the method is highly efficient and specific in labeling 8N3-NAD and 8N3-NADP, and can be applied to the identification of NAD(P) interacting proteins.
[0060] In summary, an efficient and highly specific photosensitivity strategy was developed to identify the interacting proteins of cADPR and NADP. Probe 1 only requires the addition of an azide modification to the 8-position adenine of the ligand to form a photosensitive reactive group, largely preserving the original ligand's chemical structure and properties. Furthermore, an EBT probe was designed and synthesized, introducing a reporter group onto the probe via a one-step enzymatic reaction, increasing labeling specificity and reducing experimental complexity. Therefore, this labeling method can be applied to subsequent studies of complex biological samples.
[0061] Figure 4These are the results of 8N3-NADP-labeled CD38(E226Q). Figure A shows the Western blot results after labeling with 1 μM 8N3-NAD, 0.5 μg CD38(E226Q), and 10 μg BSA; Figure B shows the Western blot results after labeling with 1 μM 8N3-NADP, 0.5 μg CD38(E226Q), and 10 μg BSA. NAD(P): 50 μM, EBT: 200 μM, Strep-HRP: for detecting biotin signals.
[0062] II. Identification of cADPR interacting proteins in cell lysates
[0063] The workflow for identifying interacting proteins using probes designed in this experiment is as follows: Figure 5 As shown, this method is named Enzyme-coupled Photolabeling Mass Spectrometry (EPL-MS). Figure 5 As shown, firstly, cell lysate was incubated with a photoaffinity probe to allow the target protein and probe to bind. Simultaneously, control and competitive groups were set up, one without probe and the other with protoligand. Photocrosslinking labeling was performed under 254 nm irradiation to covalently bind the target protein and probe. Protein denaturation was achieved through SDS and heat treatment, fully exposing the probe bound in the protein pocket. To avoid interference from excess probe and SDS on the enzymatic reaction, excess SDS and probe were removed by buffer replacement. Next, the reaction substrate EBT was added, and the affinity tag was linked to the probe under the catalysis of cyclase (E98G). Then, SDS treatment was added to denature the cyclase (E98G) and terminate the reaction. Excess EBT was then removed by buffer replacement. The target protein was enriched with streptavidin, and the enriched proteins were identified by protein gel staining, Western blotting, and proteomics. Finally, candidate cADPR and NAD(P) interacting proteins were obtained by analyzing the proteomic data.
[0064] Figure 5 The workflow for identifying interacting proteins using EPL-MS includes introducing an azide group onto the adenine of the ligand to form a photosensitive reactive group, photocrosslinking followed by biotin linkage via an exchange reaction; purification with streptavidin; and identification of the labeled protein using Western blot and proteometry techniques.
[0065] Protein labeling experiments were performed using digitalis saponins or RIPA lysis buffer to extract cytosolic proteins and total proteins from HEK293 and Ins-1E cells. Results are as follows: Figure 6 As shown, Figure 6The results showed that in Ins-1E cytoplasmic proteins, the addition of the 8N3-cADPR probe significantly labeled a protein of approximately 75 kDa, while almost no labeling signal was detected in the probe-free group and the cADPR competition group. No labeling signal was detected in HEK293 cytoplasmic proteins. When using total Ins-1E protein as the labeled sample, the results were as follows... Figure 7 As shown, no specific labeled bands were observed by Western blot. This is mainly because the abundance of endogenous biotinylated proteins in the total protein mass affects the detection of biotinylated labeled proteins in vitro. After enriching the labeled proteins with streptavidin, they were identified by proteomic methods. Figure 8 The results showed that the specifically labeled 75kDa protein was SARM1. 13 and 34 SARM1 peptides were detected in the cytoplasmic protein and total protein profiles labeled with 8N3-cADPR, respectively. In contrast, almost no SARM1 signal was detected in the probe-free group and the cADPR competition group, indicating that SARM1 can be specifically labeled by 8N3-cADPR. To further verify the mass spectrometry identification results, dN-SARM1 overexpressed in HEK293 cells was extracted, purified via affinity chromatography, and then used for labeling experiments. Figure 9 The results showed that dN-SARM1 could be specifically labeled by 8N3-cADPR, while the labeling signal was almost undetectable in the probe-free group, the cADPR-competitive group, and the OVA protein. SARM1, as a multifunctional signaling enzyme, is activated in neurons to both synthesize and hydrolyze cADPR, thus it is also a positive cADPR-binding protein. Its expression in Ins-1E cells was discovered for the first time using EPL-MS.
[0066] Mass spectrometry results for GAPDH, identified as a cADPR-binding protein in 2017, showed that the number of peptides in the probe-added group, the probe-free group, and the cADPR-competing group were essentially the same, indicating non-specific enrichment. This suggests that GAPDH may not be a cADPR-binding protein. Besides SARM1, no other specifically labeled proteins were detected in the proteomic analysis, possibly due to the low abundance of cADPR-interacting proteins in Ins-1E cells.
[0067] Figures 6 to 9 These are the results of EPL-MS identification of cADPR interacting proteins in Ins-1E cells, among which... Figure 6 Western blot results of cytoplasmic proteins of Ins-1E and HEK293 cell lines labeled with 5 μM 8N3-cADPR, cADPR: 250 μM; Figure 7Western blot results of total protein in the Ins-1E cell line labeled with 5 μM 8N3-cADPR. The labeled protein was enriched using streptavidin. Input: before enrichment, IP: after enrichment; cADPR: 200 μM. Figure 8 To analyze the number of SARM1 and GAPDH peptides detected in 8N3-cADPR-labeled Ins-1E by mass spectrometry; Figure 9 The results of Western blot analysis were obtained after labeling 5 μM 8N3-cADPR with immunoprecipitated dN-SARM1 and 15 μg OVA.
[0068] These results indicate that EPL-MS can specifically identify cADPR interacting proteins in complex biological samples. SARM1, a known cADPR-binding protein, was shown to be expressed in Ins-1E cells for the first time, while cADPR has been shown to be associated with insulin secretion; whether SARM1 is involved in this process requires further investigation.
[0069] III. Identification of NAD(P) interacting proteins in cell lysates
[0070] This experiment selected cytoplasmic proteins from the HEK293 cell line to study NAD(P) interacting proteins. The results are as follows: Figure 10 As shown, in the 8N3-NAD labeling, two proteins were specifically labeled, with sizes of approximately 55 kDa and 32 kDa, respectively. In the 8N3-NADP labeling, three proteins were specifically labeled, with sizes of approximately 70 kDa, 60 kDa, and 37 kDa, respectively. Analysis of the labeled characteristic proteins revealed that although 8N3-NAD and 8N3-NADP have very similar structures, the labeling results show significant differences, indicating that these two probes have different selectivities for the proteins, which is consistent with the actual situation of protoligands.
[0071] Figure 10 Western blot results for 8N3-NAD(P)-labeled cytoplasmic proteins of HEK293 cells, including 1 μM 8N3-NAD(P)-labeled NAD(P) interacting proteins in the cytoplasmic proteins of HEK293 cells, NAD(P): 50 μM, Strep-HRP: detecting biotin signal.
[0072] After enrichment with streptavidin, the labeled proteins were identified by proteometry. In mass spectrometry identification, proteins with 1.5 times more polypeptides in the probe group than in the probe-free group and the NAD(P) competition group were considered to be 8N3-NADP-specific labeled proteins. Cluster analysis of the 8N3-NAD-specific labeled proteins yielded the following results: Figure 11 As shown, Figure 11The results showed that these were NAD-binding proteins, ATP-binding proteins, RNA-binding proteins, cell cycle-related proteins, cytoskeleton-related proteins, and ubiquitination pathway-related proteins, of which known NAD-binding proteins accounted for 4.6%. The 8N3-NADP-specific marker proteins were mainly NADP-binding proteins, ATP-binding proteins, RNA-binding proteins, and proteasome-related proteins, of which known NADP-binding proteins accounted for 17%, such as... Figure 11 As shown.
[0073] Figure 11 This is a cluster analysis of 8N3-NADP-specific labeled proteins. A protein is considered an 8N3-NADP-specific labeled protein if its number of peptides in the probe group is 1.5 times greater than that in the probe-free group and the NAD(P) competition group. Cluster analysis of these proteins was performed using the DAVID bioinformatics website. Figure A shows the 8N3-NADP-specific labeled protein clusters, mainly including 6 groups, with 38 proteins remaining unclassified; Figure B shows the 8N3-NADP-specific labeled protein clusters, mainly including 4 groups, with 39 proteins remaining unclassified. The asterisks in the figures indicate that the protein is a kinase, and the right side shows the size of the circles corresponding to different peptide numbers, as well as the p-value of the cluster analysis.
[0074] In summary, the EPL-MS method can identify multiple known NAD(P) binding proteins in cell lysates, demonstrating that this method is feasible for identifying NAD(P) interacting proteins in complex biological samples.
[0075] IV. Conclusion
[0076] To advance research on the cADPR and NAD(P) signaling pathways, this experiment developed an enzyme-coupled photoaffinity labeling-based protein spectroscopy (EPL-MS) method to identify novel interacting proteins and elucidate the functions and mechanisms of action of these three molecules.
[0077] First, a photoreactive probe was designed and synthesized by adding an azide to the 8-position of the protoligand adenine to form a photoreactive group, which largely preserved the molecular structure and properties. A biotin-containing exchangeable probe (EBT) was also designed and synthesized, and biotin was introduced onto the probe via a one-step reaction catalyzed by cyclase (E98G). Labeling experiments on recombinant proteins showed that this method has high labeling efficiency and specificity, and can specifically label the known cADPR / NAD(P) binding protein CD38 (E226Q) and cyclase in in vitro recombinant protein systems.
[0078] EPL-MS was developed by combining this labeling method with proteomic identification. Using this method, the recently discovered cADPR synthase SARM1 was identified in Ins-1E cells using the 8N3-cADPR probe. Its function in Ins-1E cells still needs to be studied. Several known NAD(P) binding proteins were also identified in HEK293 cells using the 8N3-NAD(P) probe, indicating that the method is feasible.
[0079] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of this application, and all such modifications or substitutions should be considered within the scope of protection of this application.
Claims
1. A probe for studying the working mechanism of NAD or its metabolites, characterized in that: The probe is a dual-probe combination, which includes a first probe and a second probe. The first probe is an azide-modified nicotinamide adenine dinucleotide or an azide-modified nicotinamide adenine dinucleotide metabolite, that is, an azide group is introduced at the 8-position of adenine in nicotinamide adenine dinucleotide or the nicotinamide adenine dinucleotide metabolite, so that the azide group and the adenine of nicotinamide adenine dinucleotide or the nicotinamide adenine dinucleotide metabolite itself form a photocrosslinking reactive group. The second probe is an EBT probe with biotin, and the structural formula of the EBT probe is shown in Formula 1.
2. The probe according to claim 1, characterized in that: The azido-modified nicotinamide adenine dinucleotide metabolite is azido-modified NADP, azido-modified cADPR, or azido-modified NAADP.
3. The probe according to claim 2, characterized in that: The azide-modified NADP is shown in Formula 2; Preferably, the azide-modified cADPR is as shown in Formula 3; Preferably, the azide-modified NAADP is as shown in Formula 4; 4. The probe according to any one of claims 1-3, characterized in that: The azide-modified nicotinamide adenine dinucleotide is shown in Formula 5; 5. The use of the probe according to any one of claims 1-4 in the detection of interacting proteins of NAD or its metabolites, or in the study of the working mechanism of NAD or its metabolites.
6. A method for studying the working mechanism of NAD or its metabolites using the probe described in any one of claims 1-4.
7. The method according to claim 6, characterized in that: The process includes first covalently crosslinking the first probe with the interacting protein using photocrosslinking reactive groups; then, using ADP-ribose cyclase to exchange the second probe onto the first probe; and finally, using streptavidin to identify the interacting protein through co-immunoprecipitation, Western blotting, or proteomic analysis.
8. The method according to claim 7, characterized in that: The first probe is covalently cross-linked with the interacting protein by photocrosslinking reactive groups, specifically by covalently cross-linking the photocrosslinking reactive groups of the first probe with the interacting protein under ultraviolet light catalysis.
9. A method for detecting interacting proteins of NAD or its metabolites using the probe described in any one of claims 1-4.
10. The method according to claim 9, characterized in that: The process includes first covalently crosslinking the first probe with the interacting protein using photocrosslinking reactive groups; then, using ADP-ribose cyclase to exchange and link the second probe to the first probe; and finally, using streptavidin to identify the interacting protein through co-immunoprecipitation, Western blotting, or proteomic analysis. Preferably, the first probe is covalently cross-linked with the interacting protein by photocrosslinking reactive groups, specifically including, under ultraviolet light catalysis, covalently cross-linking the photocrosslinking reactive groups of the first probe with the interacting protein.