Aaron Elias Robinson

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naïve protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases. Graphical

Background and Aims: Methionine adenosyltransferase 1A (MAT1A) is responsible for S‐adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho‐proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. Approach and Results: We performed phospho‐proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe‐dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La‐related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5′‐terminal oligopyrimidine (TOP)‐containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1‐T449 as a SAMe‐sensitive phospho‐site of cyclin‐dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1‐overexpression–mediated increase in translation. LARP1‐T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP‐ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. Conclusions: Our results reveal a SAMe‐sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.

Recent surges in mass spectrometry-based proteomics studies demands a concurrent rise in speedy and optimized data processing tools and pipelines. Although several stand-alone bioinformatics tools exist that provide protein-protein interaction (PPI) data, we developed Protein Interaction Network Extractor (PINE) as a fully automated, user-friendly, graphical user interface application for visualization and exploration of global proteome and post-translational modification (PTM) based networks. PINE also sup-ports overlaying differential expression, statistical significance thresholds and PTM sites on functionally enriched visualization networks to gain insights into proteome-wide regulatory mechanisms and PTM-mediated networks. To illustrate the relevance of the tool, we explore the total proteome and its PTM-associated relationships in two different nonalcoholic steatohepatitis (NASH) mouse models to demonstrate different context-specific case studies. The strength of this tool relies in its ability to (1) perform accurate protein identifier mapping to resolve ambiguity, (2) retrieve interaction data from multiple publicly available PPI data-bases, and (3) assimilate these complex networks into functionally enriched pathways, ontology categories and terms. Ultimately, PINE can be used as an extremely powerful tool for novel hypothesis generation to understand underlying disease mechanisms.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography–mass spectrometry (LC–MS), whereas mass spectrometry (LC–MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and tri-methylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated non-alcoholic steatohepatitis (NASH) mouse models. We report altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.Graphical

Methionine adenosyltransferase α1 (MATα1, encoded by MAT1A) is responsible for hepatic biosynthesis of S‐adenosyl methionine, the principal methyl donor. MATα1 also act as a transcriptional cofactor by interacting and influencing the activity of several transcription factors. Mat1a knockout (KO) mice have increased levels of cytochrome P450 2E1 (CYP2E1), but the underlying mechanisms are unknown. The aims of the current study were to identify binding partners of MATα1 and elucidate how MATα1 regulates CYP2E1 expression. We identified binding partners of MATα1 by coimmunoprecipitation (co‐IP) and mass spectrometry. Interacting proteins were confirmed using co‐IP using recombinant proteins, liver lysates, and mitochondria. Alcoholic liver disease (ALD) samples were used to confirm relevance of our findings. We found that MATα1 negatively regulates CYP2E1 at mRNA and protein levels, with the latter being the dominant mechanism. MATα1 interacts with many proteins but with a predominance of mitochondrial proteins including CYP2E1. We found that MATα1 is present in the mitochondrial matrix of hepatocytes using immunogold electron microscopy. Mat1a KO hepatocytes had reduced mitochondrial membrane potential and higher mitochondrial reactive oxygen species, both of which were normalized when MAT1A was overexpressed. In addition, KO hepatocytes were sensitized to ethanol and tumor necrosis factor α–induced mitochondrial dysfunction. Interaction of MATα1 with CYP2E1 was direct, and this facilitated CYP2E1 methylation at R379, leading to its degradation through the proteasomal pathway. Mat1a KO livers have a reduced methylated/total CYP2E1 ratio. MATα1’s influence on mitochondrial function is largely mediated by its effect on CYP2E1 expression. Patients with ALD have reduced MATα1 levels and a decrease in methylated/total CYP2E1 ratio. Conclusion: Our findings highlight a critical role of MATα1 in regulating mitochondrial function by suppressing CYP2E1 expression at multiple levels.