Compositions and methods for enhancing proteasome activity
A compound and hydrate technology, applied in the field of compositions and methods for improving proteasome activity, can solve problems such as affecting the degradation rate of substrates
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Embodiment 1
[0505] Embodiment 1--the synthesis of inhibitor
[0506] Figure 29 One method of preparing the pyrroles of the invention is described. Similar oxadiazole compounds can be prepared by forming a 1,3-oxadiazole instead of an pyrrole. By varying the ring substitution on the arylamine ring 1a, or substituting alkylamines, heteroarylamines, aralkylamines, etc., a wide variety of compounds can be synthesized. Likewise, compound 1e can be reacted with a variety of nucleophiles to provide a variety of compounds. provided below Figure 29 The experimental procedures for the corresponding compounds are shown.
[0507] Synthesis of 1-(4-chlorophenyl)-2,5-dimethylpyrrole (1c). The mixture of 1a (7.65g, 60.0mmol) and 1b (34.2g, 300.0mmol) was heated to 100°C in glacial acetic acid (40mL) for 1 hour, then the solvent was evaporated to dryness, and the residue was purified by silica gel column chromatography to obtain 1c (11.07g , yield: 89.8%).
[0508] Synthesis of 2-chloro-1-[1-(4-...
Embodiment 2
[0510] Example 2 - Usp14 mediates deubiquitination of substrates
[0511] To test whether Usp14 is a potent inhibitor of the human proteasome, a purification procedure was developed to generate proteasomes lacking detectable levels of the deubiquitinating enzyme Usp14 (modified from Wang et al., (2007), Biochemistry, 46, 3553-3565). Briefly, human proteasomes were large-scale affinity purified from a stable HEK293 cell line harboring HTBH-tagged hRpn11. Cells were lysed in a Dunes homogenizer using lysis buffer containing protease inhibitors (50 mM NaH 2 PO 4 [pH 7.5], 100mM NaCl, 10% glycerol, 5mM MgCl 2 , 0.5% NP-40, 5mM ATP, and 1mM DTT). Washed lysates were incubated overnight at 4°C on NeutrAvidin agarose resin (Thermo Scientific). Subsequently, the beads were washed with excess lysis buffer, followed by wash buffer (50 mM Tris-HCl [pH 7.5], 1 mM MgCl 2 and 1mM ATP). To generate VS-proteasomes, 1 to 1.5 μM Ub-VS (Boston Biochem) was added to the resin and incubated...
Embodiment 3
[0516] Example 3 - Usp14 inhibits proteasomal degradation
[0517] The effect of Usp14 on the degradation of ubiquitinated substrates was examined in an in vitro degradation assay using the ubiquitin-dependent proteasome substrate polyubiquitinated cyclin B (Ub n -ClnB). In these experiments, Ub n -ClnB was co-incubated with human proteasome (4nM) containing wild-type or catalytically inactive Usp14 (60nM). The catalytically inactive Usp used in these experiments was Usp14-C114A, which contains a mutation in the active deubiquitinating site of Usp14. Notably, both wild-type Usp14 and Usp14-C114A were able to bind to mammalian proteasome 26S ( figure 2 ). Such as Figure 5As shown, Usp14 strongly inhibits the degradation of cyclin B, while the active site mutant of Usp14 has no inhibitory effect. Active site mutants deficient for Ub n -Inhibition of ClnB degradation suggests that the activity of wild-type Usp14 to trim ubiquitin chains is required for Usp14 to inhibit p...
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