Biology subjects

Keshishian, H.

Publications and source records attributed to Keshishian, H..

27 records · Page 2Linked to original sources

bioRxiv2022

Deep proteomics identifies shared molecular pathway alterations in synapses of schizophrenia and bipolar disorder patients and mouse model

Synaptic dysfunction is implicated in the pathophysiology of schizophrenia (SCZ) and bipolar disorder (BP). We used quantitative mass-spectrometry to carry out deep and unbiased profiling of the proteome of synapses purified from the dorsolateral prefrontal cortex of 35 cases of SCZ, 35 cases of BP, and 35 controls. Compared to controls, SCZ and BP synapses showed substantial and similar proteomic alterations. Network and gene set enrichment analyses revealed upregulation of proteins associated with autophagy and certain vesicle transport pathways, and downregulation of proteins related to synaptic, mitochondrial, and ribosomal function in the synapses of individuals with SCZ or BP. Some of the same pathways (e.g., upregulation of vesicle transport, downregulation of mitochondrial and ribosomal proteins) were similarly dysregulated in the synaptic proteome of mutant mice deficient in Akap11, a recently discovered shared risk gene for SCZ and BP. Our work provides novel biological insights into molecular dysfunction at the synapse in SCZ and BP and serves as a resource for understanding the pathophysiology of these debilitating neuropsychiatric disorders.

neuroscience↗

bioRxiv2022

Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

biochemistry↗

bioRxiv2021

TAILS identifies candidate substrates and biomarkers of ADAMTS7, a therapeutic protease target in coronary artery disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease (CAD). ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating CAD; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EMEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease related biological substrates and facilitate activity-based ADAMTS7 biomarker development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/469331v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1520ad4org.highwire.dtl.DTLVardef@144f148org.highwire.dtl.DTLVardef@13cdbc1org.highwire.dtl.DTLVardef@7f7b4f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2021

A Bidirectional Switch in the Shank3 Phosphorylation State Biases Synapses toward Up or Down Scaling

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale, quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the ASD-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were hypo-phosphorylated during scaling up and hyper-phosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ S1615 of Shank3 enables scaling up or down, respectively. Collectively our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds, and can bias synapses toward upward or downward homeostatic plasticity.

neuroscience↗

bioRxiv2021

Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples

Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.

biochemistry↗

bioRxiv2020

Highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug-treatment in cells and tissues but are presently lacking. Here we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response and resistance to therapy.

cancer biology↗

bioRxiv2020

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.

microbiology↗

bioRxiv2020

Thousands of novel unannotated proteins expand the MHC I immunopeptidome in cancer

Tumor epitopes - peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies.

cancer biology↗

bioRxiv2019

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control requires a sequence motif in the 5 region of the porin transcript. Thus, LepA controls the abundance of specific proteins, likely through its activity during translation. ImportanceOur understanding of how ribosomes properly synthesis an entire cellular proteome, in all its complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the use of GTPases such as LepA, that have evolved to aid in specific protein synthesis.

microbiology↗