Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches.
Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches. Male C57BL/6J (B6) and CAST/EiJ (CAST) mice were purchased from The Jackson Laboratories (Bar Harbor, Maine) and housed in an environmentally controlled vivarium at the University of Wisconsin Biochemistry Department. Mice were provided standard rodent chow (Purina no. 5008) and water ad libitum, and maintained on a 12-hour light/dark cycle (6 AM – 6 PM). At 10 weeks of age, mice were sacrificed by CO2 asphyxiation. All animal procedures were preapproved by the University of Wisconsin Animal Care and Use Committee.
Project description:PhosphoSitePlus(®) (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330,000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1,000,000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The 'Regulatory sites' dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The 'PTMVar' dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25,000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites.
Project description:A few protein PTMs, such as phosphorylation and ubiquitination, are known to be critical in regulation of protein kinase activities. However, the roles of other PTMs have not been extensively studied in kinases. Development of a comprehensive description of all types of PTMs and discovering novel in vivo PTMs in low abundance represent major analytical challenges. Toward this goal, we have developed a strategy for systematic and accurate identification of the full-spectrum of PTMs in yeast protein kinases. Our strategy involves isolation of GST-fused kinase proteins, MS analysis, and unrestrictive PTM identification by PTMap algorithm. Among the 30 purified yeast kinases, we identified 27 different types of PTMs, and 53 PTM sites, among which are 13 novel mass shifts that have not been previously reported, likely representing novel PTMs. These results represent a significant expansion of our current understanding of PTMs in kinases and suggest highly complex regulation of kinase function.
Project description:Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, characterized by severe muscle and fat loss and yet, there are no biomarkers identified for this debilitating condition. Our objective was to identify circulating protein biomarkers using serum for human PDAC cachexia and understand their biological functions. Serum from 30 patients with PDAC was collected and protein profiles were generated using SOMAscan. The protein profiles were correlated with clinical variables such as Cancer associated weight loss (CAWL), body composition measurements of skeletal muscle index (SMI), skeletal muscle density (SMD), total adipose index (TAI) using Spearman’s correlation. Overall, 110 proteins of 1294 correlated with these clinical measures - 47 proteins for CAWL, 19 for SMI, 14 for SMD, and 30 for TAI (r-value 0.5, p<0.05). LYVE1, a homolog of CD44 implicated in tumor metastasis, was the top CAWL-associated protein (r= 0.67, p=0.0001). Protein co-expression network analysis identified immune system related pathways such as B-cell signaling, natural killer cell signaling, IL6 signaling in addition to identifying other known pathways in cachexia. Taken together, these data identify both immune system molecules and additional secreted factors and pathways not previously associated with PDAC and confirm the activation of previously identified pathways.
Project description:Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington's disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.
Project description:The formation of atherosclerotic plaques is one of the main sources of cardiovascular disease. In addition to known risk factors such as dyslipidemia, diabetes, obesity, and hypertension, endothelial dysfunction has been shown to play a key role in the formation and progression of atherosclerosis. Peroxisome proliferator-activated receptor-gamma (PPARγ), a transcription factor belonging to the steroid superfamily, is expressed in the aorta and plays a critical role in protecting endothelial function. It thereby serves as a target for treating both diabetes and atherosclerosis. Although many studies have examined endothelial cell disorders in atherosclerosis, the role of PPARγ in endothelial dysfunction is still not well understood. In this review, we summarize the possible mechanisms of action behind PPARγ regulatory compounds and post-translational modifications (PTMs) of PPARγ in the control of endothelial function. We also explore the potential use of endothelial PPARγ-targeted agents in the prevention and treatment of atherosclerosis.
Project description:Mycobacterium tuberculosis (Mtb) antigen-specific cellular response is promising for detectionof Mtb infection, but not efficient for diagnosis of TB. We firstly identified 16 TB disease-specific protein markers measured in the culture supernatant of Mtb-stimulated whole blood using a 640 human proteins array, the highest throughput antibody-based protein array available at the time when we did this study. Potential TB-related proteins were then analyzed across three different patient cohorts comprised of healthy controls, LTBI, non-TB pneumonia, and TB patients to evaluate how the biomarkers performed in diagnosing TB in the real clinical setting. The data finally reveal an eight-protein biosignature of TB.
Project description:CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3'GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3'GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3'GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3'GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3'GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.