Browsing by Subject "post-translational modification"

Now showing 1 - 5 of 5
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    Characterization of proteoforms with unknown post-translational modi cations using the MIScore
    (ACS, 2016) Kou, Qiang; Zhu, Binhai; Wu, Si; Ansong, Charles; Tolić, Nikola; Paša-Tolić, Ljiljana; Liu, Xiaowen; Department of Biohealth Informatics, School of Informatics and Computing
    Various proteoforms may be generated from a single gene due to primary structure alterations (PSAs) such as genetic variations, alternative splicing, and post-translational modifications (PTMs). Top-down mass spectrometry is capable of analyzing intact proteins and identifying patterns of multiple PSAs, making it the method of choice for studying complex proteoforms. In top-down proteomics, proteoform identification is often performed by searching tandem mass spectra against a protein sequence database that contains only one reference protein sequence for each gene or transcript variant in a proteome. Because of the incompleteness of the protein database, an identified proteoform may contain unknown PSAs compared with the reference sequence. Proteoform characterization is to identify and localize PSAs in a proteoform. Although many software tools have been proposed for proteoform identification by top-down mass spectrometry, the characterization of proteoforms in identified proteoform–spectrum matches still relies mainly on manual annotation. We propose to use the Modification Identification Score (MIScore), which is based on Bayesian models, to automatically identify and localize PTMs in proteoforms. Experiments showed that the MIScore is accurate in identifying and localizing one or two modifications.
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    Dissecting the Role of Novel O-GlcNAcylation of NF-κB in Pancreatic Cancer
    (2024-06) Motolani, Aishat Abiola; Lu, Tao; Safa, Ahmad; Dong, Charlie; Pollok, Karen; Corson, Timothy
    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited PDAC cellular proliferation, migration, and anchorage-independent growth compared to WT-p65. We also show that inhibition of NF-κB O-GlcNAcylation may mitigate gemcitabine resistance and enhance its efficacy in PDAC cells. Collectively, our study uncovers a novel aspect of NF-κB regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer.
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    Intrinsically disordered proteins link alternative splicing and post-translational modifications to complex cell signaling and regulation
    (Elsevier, 2018) Zhou, Jianhong; Zhao, Suwen; Dunker, A. Keith; Biochemistry and Molecular Biology, School of Medicine
    Intrinsically disordered proteins and regions (IDPs and IDRs) lack well-defined tertiary structures, yet carry out various important cellular functions, especially those associated with cell signaling and regulation. In eukaryotes, IDPs and IDRs contain the preferred loci for both alternative splicing (AS) and many post-translational modifications (PTMs). Furthermore, AS and/or PTMs at these loci generally alter the signaling outcomes associated with these IDPs or IDRs, where the functional cooperation of these three features is named the IDP-AS-PTM toolkit. However, the prevalence of such functional modulations remains unknown. Also, the signal-altering mechanisms by which AS, and PTMs modulate function and the extent to which AS and PTMs collaborate in their signaling modulations have not been well defined for particular protein examples. Here we focus on three important signaling and regulatory IDR-containing protein families in humans, namely G-protein coupled receptors (GPCRs), which are transmembrane proteins, the nuclear factors of activated T-cells (NFATs), which are transcription factors (TFs), and the Src family kinases (SFKs), which are signaling enzymes. The goal here is to determine how AS and PTMs individually alter the outcomes of the signaling carried out by the various IDRs and to determine whether AS and PTMs work together to bring about differential cellular responses. We also present data indicating that a wide range of other signaling IDPs or IDRs undergo both AS- and PTM-based modifications, suggesting that they, too, likely take advantage of signal outcome modulations that result from collaboration between these two events. Hence, we propose that the widespread cooperation of IDPs, AS and/or PTMs provides a IDP-AS-PTM toolkit and substantially contributes to the vast complexity of eukaryotic cell signaling systems.
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    Partially Penetrant Cardiac Neural Crest Defects in Hand1 Phosphomutant Mice: Dimer Choice That Is Not So Critical
    (Springer, 2019-07-23) Firulli, Beth A.; Firulli, Anthony B.; Pediatrics, School of Medicine
    Hand1 is a basic Helix-loop-Helix transcription factor that exhibits post-translationally regulated dimer partner choice that allows for a diverse set of Hand1 transcriptional complexes. Indeed, when Hand1 phosphoregulation is altered, conditionally activated hypophorylation (Hand1PO4−) and phosphorylation mimic (Hand1PO4+) Hand1 alleles disrupt both craniofacial, and limb morphogenesis with 100% penetrance. Interestingly, activation of conditional Hand1 Phosphomutant alleles within post migratory neural crest cells produce heart defects that include ventricular septal defects, double outlet right ventricle, persistent truncus arteriosus with partial penetrance. Single vs double lobed thymus is a distinguishing feature between Wnt1Cre;Hand1PO4−/+ and Wnt1-Cre;Hand1PO4+/+ mice. These data show that although Hand1 dimer regulation play critical and consistent roles in disrupting craniofacial and limb morphogenesis, Hand1 dimer regulation during cardiac outflow track formation is less critical for normal morphogenesis. This review will present the OFT phenotypes observed in Hand1 Phosphomutant mice, and discuss possible mechanisms of how penetrance differences within the same tissues within the same embryos could be variable.
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    Regulation of Protein Arginine Methyl Transferase 5 by Novel Serine 15 Phosphorylation in Colorectal Cancer
    (2020-01) Hartley, Antja-Voy Anthoneil; Lu, Tao; Harrington, Maureen; Pollock, Karen; Safa, Ahmad; Yamamoto, Bryan
    The overexpression of protein arginine methyltransferase 5 (PRMT5) is strongly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Previously, we demonstrated that PRMT5 overexpression could substantially augment activation of NF-κB via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect on the transcriptional competence of p65. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. We report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A-PRMT5), in either HEK293 cells or HT29, DLD1 and HCT116 CRC cells attenuated NF-κB activation compared to wild type (WT)-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, we found that overexpression of S15A-PRMT5 mutant attenuated the expression of a subset of NF-κB target genes through decreased p65 occupancy at their respective promoters. Importantly, the S15A-PRMT5 mutant also reduced IL-1β-induced methyltransferase activity of PRMT5 as well as its ability to form a complex with p65. Finally, we observed that the S15A-PRMT5 mutant diminished the growth, migratory and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, our findings provide strong evidence that novel phosphorylation of PRMT5 at S15 is critical to its regulation of NF-κB and plays an essential role in promoting the cancer-associated functions exerted by the PRMT5/NF-κB axis. Therefore, development of inhibitors to block phosphorylation of PRMT5 at S15 could become a potential novel therapeutic approach to treat CRC.