Similar Datasets
Project description:To check whether mRNA level change for the changed proteins in protein level.
2020-11-01 | GSE156101 | GEO
Project description:Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most compressively studied DUBs. Since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC and PTEN, USP7 becomes a promising drug target. However, systematic identification of USP7 substrates have not yet been performed. In this study, we carried out proteome profiling with label-free quantification using control and single/double KO cells of USP7 and its closest homolog USP47. Our proteome profiling for the first time reveal the proteome changes caused by USP7 and/or USP47 deletion. Combining protein profiling, transcriptome analysis and tandem-affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates, which include known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. Further, we showed that MGA deletion partially phenocopied proliferation defect observed in cells with USP7 deletion. Additionally, we established that USP7 deletion repress interferon gamma response. Moreover, we showed that the cytotoxicity of USP7 inhibitors were independent of USP7. In conclusion, our study is the first proteome-wide analysis of potential USP7 substrates, which provides a resource for further functional studies.
2021-08-04 | MSV000087940 | MassIVE
Project description:Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.
| S-EPMC8052291 | biostudies-literature
Project description:Neuraminidases (sialidases) are enzymes that hydrolytically remove sialic acid from sialylated proteins and lipids. Neuraminidases are encoded by a range of human pathogens, including bacteria, viruses, fungi, and protozoa. Many pathogen neuraminidases are virulence factors, indicating that desialylation of host glycoconjugates can be a critical step in infection. Specifically, desialylation of host cell surface glycoproteins can enable these molecules to function as pathogen receptors or can alter signaling through the plasma membrane. Despite these critical effects, no unbiased approaches exist to identify glycoprotein substrates of neuraminidases. Here, we combine previously reported glycoproteomics methods with quantitative proteomics analysis to identify glycoproteins whose sialylation changes in response to neuraminidase treatment. The two glycoproteomics methods-periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL)-rely on chemoselective labeling of sialylated and nonsialylated glycoproteins, respectively. We demonstrated the utility of the combined approaches by identifying substrates of two pneumococcal neuraminidases in a human cell line that models the blood-brain barrier. The methods deliver complementary lists of neuraminidase substrates, with GAL identifying a larger number of substrates than PAL (77 versus 17). Putative neuraminidase substrates were confirmed by other methods, establishing the validity of the approach. Among the identified substrates were host glycoproteins known to function in bacteria adherence and infection. Functional assays suggest that multiple desialylated cell surface glycoproteins may act together as pneumococcus receptors. Overall, this method will provide a powerful approach to identify glycoproteins that are desialylated by both purified neuraminidases and intact pathogens.
| S-EPMC4838540 | biostudies-literature
Project description:Turnover of cellular proteins is regulated by Ubiquitin Proteasome System (UPS). Components of this pathway, including the proteasome, ubiquitinating enzymes and deubiquitinating enzymes, are highly specialized and tightly regulated. In this mini-review we focus on the de-ubiquitinating enzyme USP7, and summarize latest advances in understanding its structure, substrate specificity and relevance to human cancers. There is increasing interest in UPS components as targets for cancer therapy and here we also overview the recent progress in the development of small molecule inhibitors that target USP7.
| S-EPMC6481172 | biostudies-literature
Project description:Transcriptome analysis of total RNA samples from HEK293-PIGS-KO and HEK293-PIGS-UBE2J1-DKO cells. To check whether KO of UBE2J1 upregulates genes of GPI biosthesis pathway, we used microarrays to analyze gene expression change by KO of UBE2J1 and comfirmed that known GPI pathway genes are not changed by ERAD-deficiency.
2020-01-08 | GSE140855 | GEO
Project description:Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.
| S-EPMC2866015 | biostudies-literature
Project description:Our lack of total understanding of the intricacies of how enzymes behave has constrained our ability to robustly engineer substrate specificity. Furthermore, the mechanisms of natural evolution leading to improved or novel substrate specificities are not wholly defined. Here we generate near-comprehensive single-mutation fitness landscapes comprising >96.3% of all possible single nonsynonymous mutations for hydrolysis activity of an amidase expressed in E. coli with three different substrates. For all three selections, we find that the distribution of beneficial mutations can be described as exponential, supporting a current hypothesis for adaptive molecular evolution. Beneficial mutations in one selection have essentially no correlation with fitness for other selections and are dispersed throughout the protein sequence and structure. Our results further demonstrate the dependence of local fitness landscapes on substrate identity and provide an example of globally distributed sequence-specificity determinants for an enzyme.
| S-EPMC5467163 | biostudies-literature
Project description:MicroRNAs (miRNAs) play an important role in carcinogenesis by regulating the protein translation from their targeted messenger RNAs (mRNA). Aristolochic Acid (AA) is a potent carcinogen that can induce kidney tumors in both human and experimental animals. Although the expression and roles of miRNAs have been extensively studied in cancer tissues or tissues treated by carcinogens, it is still unclear how the three levels of gene expression, including miRNA, mRNA and protein, are regulated and coordinated during the early stage of carcinogenesis. Here, we treated rats with 10 mg/kg AA or vehicle control for 12 weeks and the kidney tissues were quickly isolated and frozen immediately after the treatment. Eight kidney samples (4 for the treatment and 4 for the control) were used for analyzing miRNA and mRNA expressions with deep sequencing, and protein expressions with proteomics. MiRNA expressions were significantly changed by AA treatment. The treated samples were well separated from the control samples in both principal component analysis (PCA) and hierarchical clustering analysis (HCA). Quantitative measurements of six miRNAs using TaqMan real-time PCR were consistent with the deep sequencing results in terms of both direction and magnitude of gene expression change. Sixty-three miRNAs (adjusted p value < 0.05 and fold change > 1.5), 6,794 mRNAs (adjusted p value < 0.05 and fold change > 2.0), and 800 proteins (fold change > 2.0) were significantly altered by AA treatment. By combining the results of a computational target predicting algorithm, and the mRNA and protein expression data, 143 genes were found by the differentially expressed miRNAs (DEMs). The DEM-targeted genes are mainly related to cancer, cell growth, which reflects the carcinogenic processes in the AA-treated rat kidneys. Groups of four 6 week-old male Big Blue rats were treated with AA as its sodium salt at 10.0 mg/kg body weight by gavage (4 ml/kg body weight) 5 times a week for 12 weeks. The rats that were treated with 0.9% sodium chloride on the same schedule were used as control. microRNAs were isolated from rat kidneys and subject to next-generation sequencing to determine the expression profile.
2012-01-01 | E-GEOD-33703 | biostudies-arrayexpress
Project description:Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin ?, meprin ?, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1' position because of a conserved positively charged residue in the active cleft subsite (S)1'. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2' or P3' leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1?, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing.
| S-EPMC3186203 | biostudies-literature