Biology subjects

Carr, S. A.

Publications and source records attributed to Carr, S. A..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis

O-GlcNAc transferase (OGT) catalyzes the modification of serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We employed a genome-wide CRISPR-Cas9 viability screen in mouse embryonic stem cells (mESCs) with inducible Ogt gene deletion and showed that the block in cell viability induced by OGT deficiency stems from mitochondrial dysfunction secondary to mTOR hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phosphoproteomic screen. Our study highlights a novel series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.

cell biology↗

bioRxiv2021

Cloud-based DIA data analysis module for signal refinement improves accuracy and throughput of large datasets.

Data-independent acquisition (DIA) is a powerful mass spectrometry method that promises higher coverage, reproducibility, and throughput than traditional quantitative proteomics approaches. However, the complexity of DIA data caused by fragmentation of co-isolating peptides presents significant challenges for confident assignment of identity and quantity, information that is essential for deriving meaningful biological insight from the data. To overcome this problem, we previously developed Avant-garde, a tool for automated signal refinement of DIA and other targeted mass spectrometry data. AvG is designed to work alongside existing tools for peptide detection to address the reliability and quantitative suitability of signals extracted for the identified peptides. While its use is straightforward and offers efficient refinement for small datasets, the execution of AvG for large DIA datasets is time-consuming, especially if run with limited computational resources. To overcome these limitations, we present here an improved, cloud-based implementation of the AvG algorithm deployed on Terra, a user-friendly cloud-based platform for large-scale data analysis and sharing, as an accessible and standardized resource to the wider community.

bioinformatics↗

bioRxiv2021

MONTE enables serial immunopeptidome, ubiquitylome, proteome, phosphoproteome, acetylome analyses of sample-limited tissues

Serial multiomic analyses of proteome, phosphoproteome and acetylome provides functional insights into disease pathology and drug effects while conserving precious human material. To date, ubiquitylome and HLA peptidome analyses have required separate samples for parallel processing each using distinct protocols. Here we present MONTE, a highly-sensitive multi-omic native tissue enrichment workflow that enables serial, deepscale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome and acetylome from the same tissue samples. We demonstrate the capabilities of MONTE in a proof-of-concept study of primary patient lung adenocarcinoma(LUAD) tumors. Depth of coverage and quantitative precision at each of the omes is not compromised by serialization, and the addition of HLA immunopeptidomics enables identification of putative immunotherapeutic targets such as cancer/testis antigens and neoantigens. MONTE can provide insights into disease-specific changes in antigen presentation, protein expression, protein degradation, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment.

cancer biology↗

bioRxiv2021

Novel antibodies for the simple and efficient enrichment of native O-GlcNAc modified peptides

Antibodies against post-translational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides, and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1,300 unique O-GlcNAc-modified peptides and over 1,000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

biochemistry↗

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↗

bioRxiv2021

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-{varepsilon}-GG peptides are enriched with anti-K-{varepsilon}-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-{varepsilon}-GG antibody (mK-{varepsilon}-GG) and a magnetic particle processor. We report the identification of [~]20,000 ubiquitylation sites from a TMT10-plex with 500 g input per sample processed in [~]2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

biochemistry↗

bioRxiv2021

BCOR and BCORL1 mutations disrupt PRC1.1 repressive function in leukemia by unlinking the RING-PCGF1 enzymatic core from target genes

BCOR and its paralog BCORL1 encode subunits of the Polycomb repressive complex 1.1 (PRC1.1) and are recurrently mutated in myeloid malignancies. We show that leukemia-associated BCOR/BCORL1 mutations unlink the PRC1.1 RING-PCGF enzymatic core from the KDM2B-containing chromatin targeting auxiliary subcomplex, either by causing complete protein loss or expression of a C-terminally truncated protein lacking the PCGF Ub-like fold discriminator (PUFD) domain. By uncoupling PRC1.1 repressive function from target genes, BCOR/BCORL1 mutations activate aberrant cell signaling programs that confer acquired resistance to treatment. This study provides a mechanistic basis for Polycomb repressive dysfunction as a key oncogenic driver in myeloid malignancies and identifies a potential strategy for targeted therapy in BCOR-mutated cancer.

cancer biology↗

bioRxiv2021

Proximity-labeling reveals novel host and parasite proteins at the Toxoplasma parasitophorous vacuole membrane

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelopes its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this crucial interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three novel parasite proteins (TGGT1_269950, TGGT1_215360, and TGGT1_217530) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the PVM and, given that three of the validated host proteins are components of the ESCRT machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. ImportanceToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

microbiology↗

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

PANOPLY: A cloud-based platform for automated and reproducible proteogenomic data analysis

Proteogenomics involves the integrative analysis of genomic, transcriptomic, proteomic and post-translational modification data produced by next-generation sequencing and mass spectrometry-based proteomics. Several publications by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and others have highlighted the impact of proteogenomics in enabling deeper insight into the biology of cancer and identification of potential drug targets. In order to encapsulate the complex data processing required for proteogenomics, and provide a simple interface to deploy a range of algorithms developed for data analysis, we have developed PANOPLY--a cloud-based platform for automated and reproducible proteogenomic data analysis. A wide array of algorithms have been implemented, and we highlight the application of PANOPLY to the analysis of cancer proteogenomic data.

bioinformatics↗

bioRxiv2020

Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/387209v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1223baforg.highwire.dtl.DTLVardef@7c87c6org.highwire.dtl.DTLVardef@1b5e7cdorg.highwire.dtl.DTLVardef@185449b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

immunology↗

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

Biomarkers to distinguish bacterial from viral pediatric clinical pneumonia in a malaria endemic setting

BACKGROUNDDifferentiating the etiology of acute febrile respiratory illness in children is a challenge in low-income, malaria-endemic settings because the main pathogens responsible (viruses, bacteria, and malaria parasites) overlap in clinical presentation and frequently occur together as mixed infections. The critical task is to rapidly identify bacterial pneumonia to enable appropriate antibiotic treatment, ideally at point of care. Current diagnostic tests are insufficient and there is a need for the discovery and development of new tools. Here we report the identification of a unique biomarker signature that can be identified in blood samples. METHODSBlood samples from 195 pediatric Mozambican patients with clinical pneumonia were analyzed with an aptamer-based high dynamic range assay to quantify [~]1200 proteins. For discovery of new biomarkers, we identified a training set of patient samples in which the underlying etiology of the pneumonia was established as bacterial, viral or malaria. Proteins whose abundances varied significantly between patients with verified etiologies (FDR<0.01) formed the basis for predictive diagnostic models that were created using machine learning techniques (Random Forest, Elastic Net). These models were validated on a dedicated test set of samples. RESULTS219 proteins had significantly different abundances between bacterial and viral infections, and 151 differed between bacterial infections and a mixed pool of viral and malaria infections. Predictive diagnostic models achieved >90% sensitivity and >80% specificity, regardless of whether one or two pathogen classes were present. Bacterial pneumonia was strongly associated with markers of neutrophil activity, in particular neutrophil degranulation. Degranulation markers included HP, LCN2, LTF, MPO, MMP8, PGLYRP1, RETN, SERPINA1, S100A9, and SLPI. CONCLUSIONBlood protein signatures highly associated with neutrophil biology reliably differentiated bacterial pneumonia from other causes. With appropriate technology, these markers could provide the basis for a rapid diagnostic for field-based triage for antibiotic treatment of pediatric pneumonia.

genomics↗

bioRxiv2020

Proteomics of protein trafficking by in vivo tissue-specific labeling

Secreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors large-scale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors. This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states. One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteome-wide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles.

physiology↗

bioRxiv2020

CBL mutations promote activation of PI3K/AKT signaling via LYN kinase

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series of CBL mutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling in CBL-mutant cells. Furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients with CBL-mutated myeloid malignancies. Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associated CBL mutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity in CBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors in CBL-mutated myeloid malignancies.

cancer biology↗

bioRxiv2020

Mapping the unique and shared functions of oncogenic KRAS and RIT1 with proteome and transcriptome profiling

Aberrant activation of RAS oncogenes is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in [~]30% of tumors. Recently, we identified somatic mutation of the RAS-family GTPase RIT1 in lung adenocarcinoma, but relatively little is known about the biological pathways regulated by RIT1 and how these relate to the oncogenic KRAS network. Here we present quantitative proteomic and transcriptomic profiles from KRAS-mutant and RIT1-mutant isogenic lung epithelial cells and globally characterize the signaling networks regulated by each oncogene. We find that both mutant KRAS and mutant RIT1 promote S6 kinase, AKT, and RAF/MEK signaling, and promote epithelial-to-mesenchymal transition and immune evasion via HLA protein loss. However, KRAS and RIT1 diverge in regulation of phosphorylation sites on EGFR, USO1, and AHNAK proteins. The majority of the proteome changes are related to altered transcriptional regulation, but a small subset of proteins are differentially regulated by both oncoproteins at the post-transcriptional level, including intermediate filament proteins, metallothioneins, and MHC Class I proteins. These data provide the first global, unbiased characterization of oncogenic RIT1 network and identify the shared and divergent functions of oncogenic RIT1 and KRAS GTPases in lung cancer.

cancer biology↗

bioRxiv2020

Split-TurboID enables contact-dependent proximity labeling in cells

Proximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

cell biology↗