Research Articles
- Research Article
Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism
In Brief The native conformation of the protease inhibitor A2M was investigated using negative stain electron microscopy, small-angle X-ray scattering, and cross-linking mass spectrometry. The low-resolution model built from these data describes a hollow tubular configuration that explains several aspects of A2M’s unique trapping mechanism. This model was further validated by two recombinantly expressed A2M mutants, which probed the location of the bait region and demonstrated the existence of a critical interface between A2M’s disulfide-bridged dimers. - Research Article
The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia
In Brief Here, we demonstrated that the knockdown of non–T cell activation linker (NTAL) in acute myeloid leukemia (AML) cells was linked to reduced cell proliferation and survival in vitro and in vivo. In addition, we identified NTAL interactors in AML using label-free protein quantification. NTAL interactors presented a high expression in patients with AML, being associated with a leukemic granulocyte–macrophage progenitor-like state. Finally, NTAL interactors were capable to predict survival in a large subset of patients with AML. These data provide evidence that NTAL and its interactors could represent potential therapeutic targets for granulocyte–macrophage progenitor-like leukemias. - Research Article
The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development
In Brief We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing. - Technological Innovation and Resources
GAGrank: Software for Glycosaminoglycan Sequence Ranking Using a Bipartite Graph Model
In Brief We demonstrate GAGrank, an algorithm that uses a bipartite graph model for sequencing glycosaminoglycans from EDD or NETD tandem mass spectra. The process involves first assigning glycosaminoglycan product ions using the GAGfinder algorithm. The second step is to rank possible sequences using GAGrank. We show GAGrank’s ability to sequence isomeric mixtures. - Research Article
Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia
In Brief We conducted a sensitive and high-throughput reverse phase protein array to attain protein expression profiles of primary fibroblasts from patients with Friedreich's ataxia (FRDA) and unaffected controls using a pool of 217 validated antibodies. Our extensive bioinformatics analyses correlated differentially expressed (DE) proteins with critical disease parameters. Expression levels of several integrin proteins specifically associated with hearing loss in FRDA. Also, reverse phase protein array data integrated with transcriptome data uncovered defects in retinoic acid metabolism in FRDA samples. - Research Article
Adipose Triglyceride Lipase Loss Promotes a Metabolic Switch in A549 Non–Small Cell Lung Cancer Cell Spheroids
In Brief We here demonstrate that loss of adipose triglyceride lipase (ATGL) in an in-vitro model of 3D lung carcinoma cell (A549) culture leads to a metabolic switch supporting larger spheroid growth and better adaptation to hypoxia. Cancer spheroids lacking ATGL show a higher abundance of glucose transporters, increased glucose uptake and lactate production, and use alternative lipid metabolic pathways. Overall, our data suggest consideration of ATGL as potentially relevant target in lung cancer. - Research Article
Increased Wheat Protein Content via Introgression of an HMW Glutenin Selectively Reshapes the Grain Proteome
In Brief The differences between the grain proteome of three wheat cultivars and corresponding Ay HMW-GS–introgressed near-isogenic lines have been determined. In addition to increased abundance of 1Ay HMW-GS, 115 differentially expressed proteins were also discovered. This revealed that introgression of the 1Ay21∗ HMW-GS increases wheat grain protein content and improves bread-making quality in association with a wider reshaping of the grain proteome network. - Research Article
Revealing the Dynamic Allosteric Changes Required for Formation of the Cysteine Synthase Complex by Hydrogen-Deuterium Exchange MS
In Brief We have used hydrogen/deuterium exchange MS to unveil the allosteric changes occurring during complex formation of CysE and CysK, the last two enzymes of cysteine biosynthetic pathway in bacteria. Significant changes in conformation and dynamics occur in each protein upon complex formation, including long distance intraprotein and interprotein communication. Being absent in mammals, both CysE and CysK represent potential targets for new antibacterial drugs, and our results on the formation and allostery of the complex could help guide drug development. - Research Article
Proteogenomic Assessment of Intraspecific Venom Variability: Molecular Adaptations in the Venom Arsenal of Conus purpurascens
In Brief Cone snail venom is an extensive source of active molecules that have potential pharmacological and biotechnological applications. We employed a top-down functional proteogenomic approach to assess the injected venom of Conus purpurascens. The two distinct venom profiles found will interact differently to target neural pathways aimed to immobilize prey. These venom expression patterns will aid target prediction and the development of conotoxins into drug leads or neural probes. - Research Article
Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis
In Brief The molecular changes associated with normothermic ex-vivo kidney perfusion (NEVKP) compared with static cold storage were studied using discovery proteomics in a porcine model. NEVKP resulted in increased expression of mitochondrial proteins (ETFB, CPT2) responsible for critical metabolic steps of ATP-synthesis. PPARGC1A, PPARA/D, and RXRA were computationally predicted as upstream regulators of proteins increased in NEVKP and showed increased mRNA expression in NEVKP-treated kidneys. PPAR-family members and their target proteins may represent new therapeutic targets to ameliorate ischemia-reperfusion injury. - Research Article
Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins
In Brief The polyphosphoinositide (PPIn) phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) localizes to the nucleus and nucleolus. Using an affinity enrichment MS approach, the nuclear PtdIns(3,4,5)P3 interactome identified new interaction partners associated with the nucleolus. Among these, the DNA repair PARP1 protein, colocalized to the nucleolus with PtdIns(3,4,5)P3 and showed direct interaction to PPIn via three polybasic regions. The nuclear PtdIns(3,4,5)P3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5)P3-mediated interactions in the nucleus and nucleolus. - Research Article
Deeply Mining a Universe of Peptides Encoded by Long Noncoding RNAs
In Brief This study proposed a new and effective strategy for the improved discovery and identification of novel SEPs, including the construction of databases maximally collecting all putative small ORFs from human and mouse lncRNA transcripts in NONCODE and the effective enrichment of polypeptides based on 30-kDa molecular weight cutoff (MWCO) membrane and C8 solid-phase extraction column. This effort led to the discovery of 762 novel lncRNA-encoded SEPs from multiple cell lines and tissues. - Research Article
Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients
In Brief Neutrophils contribute to the extracellular DNA pool by forming neutrophil extracellular traps (NETs), which cause sputum thickening, pulmonary inflammation, and hindrance to gaseous exchange during infections. Here, we demonstrate the presence of NETs in sputum from severe COVID-19 patients using mass spectrometry and immunofluorescence analyses. Treatment with clinically approved recombinant human DNase reduced NETs and was associated with improved recovery and reduced inflammation. Targeting NETs using DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies. - Research Article
Age-Associated Proteomic Signatures and Potential Clinically Actionable Targets of Colorectal Cancer
In Brief The incidence of early-onset colorectal cancer (CRC) has been increasing since 1990s, whereas the overall CRC frequency is declining. The underlying mechanisms of age-related clinical differences remain unknown. Here, we reported the proteomic signatures of CRC across age groups. Lots of proteins with adjusted intensities significantly correlated with age. Some proteins were verified as potential clinically actionable targets. This study identifies age-associated proteomic signatures and potential therapeutic targets of CRC and helps to make a precise decision on CRC treatment. - Research Article
Proteome Characterization of Glaucoma Aqueous Humor
In Brief Liu et al. characterized the proteome of aqueous humor from three types of glaucoma. Fifty-seven primary acute angle-closure glaucoma (PAACG), 50 primary chronic angle-closure glaucoma (PCACG), 35 neovascular glaucoma (NVG), and 33 cataract patient samples were analyzed using data-independent analysis and parallel reaction monitoring. Lipid metabolism, immune response, and cell death pathways showed different degrees of activation among the three types of glaucoma but were all higher relative to cataract. SERPIND1 was discovered as a vital protein in glaucoma. - Research Article
A Reductionist Approach Using Primary and Metastatic Cell–Derived Extracellular Vesicles Reveals Hub Proteins Associated with Oral Cancer Prognosis
In Brief A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 ‘hub proteins’ that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer. - Research Article
Comparative Host Interactomes of the SARS-CoV-2 Nonstructural Protein 3 and Human Coronavirus Homologs
In Brief SARS-CoV-2 nonstructural protein 3 (nsp3) facilitates virion biogenesis and modulates host ubiquitinylation/ISGylation. The SARS-CoV-2 nsp3 host interactome has not been fully characterized. Using affinity purification–mass spectrometry, we identify interactors of SARS-CoV-2 nsp3 and homologs from four CoV strains. We show the N-terminus of SARS-CoV-2 nsp3 interacts with the transcription factor ATF6 and suppresses its stress response. This work examines the interface between a key CoV protein and host cells, highlighting potential dependencies for antiviral therapeutics. - Research Article
Proteomic Landscape of Exosomes Reveals the Functional Contributions of CD151 in Triple-Negative Breast Cancer
In Brief Using a quantitative proteomics approach, Li et al. characterized the proteomes of triple-negative breast cancer (TNBC) patient–derived serum exosomes and found the tetraspanin CD151 to be significantly enriched. Proteomic analysis of CD151-deleted exosomes and cells showed regulation of ribosomal and complement protein secretion. CD151-deleted exosomes were shown to significantly decrease the migration and invasion of TNBC cells, indicating that exosomal CD151 may be a potential therapeutic target for TNBC. - Research Article
Unbiased Proteomic and Phosphoproteomic Analysis Identifies Response Signatures and Novel Susceptibilities After Combined MEK and mTOR Inhibition in BRAFV600E Mutant Glioma
In Brief BRAFV600E is a key oncogenic driver in glioma, melanoma, and colon cancer. These tumors escape mitogen-activated protein kinase pathway inhibition by upregulating mammalian target of rapamycin signaling. Using comprehensive unbiased proteomic and phosphoproteomic analysis of an in vivo BRAFV600E mutant glioma model treated with inhibitors of both these key pathways, we characterize the tumor and stromal response and suggest additional therapeutic targets for BRAF-driven cancers, including epidermal growth factor receptor and class 1 histone deacetylases. - Research Article
Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network
In Brief Various pathologies including neurodegenerative diseases and cancers result from disruptions to or stress of ER homeostasis. One key protein proposed to act in quality control processes maintaining ER homeostasis is EDEM2. Using affinity proteomics and sucrose-density sedimentation, we identified several new EDEM2 partners involved in quality control. Moreover, we defined novel endogenous candidates for EDEM2-dependent degradation by combining glycoproteomics and SILAC-based proteomics. Our data suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously thought. - Research Article
Proteomics, Lipidomics, Metabolomics, and 16S DNA Sequencing of Dental Plaque From Patients With Diabetes and Periodontal Disease
In Brief The human oral cavity houses a complex ecosystem of microbes, some of which have pathogenic influence on the host. Multi-omics analysis of oral plaques revealed key players in microbial communities derived from diabetic and periodontal disease patients. With cross-omic correlation analysis, we found host-specific proteins and associated lipids that were elevated in plaques from periodontal disease patients. Furthermore, this multi-omic approach leads to the finding that oral community member Lautropia mirabilis synthesizes monomethyl phosphatidylethanolamine, an uncommon lipid in oral microbiota. - Research Article
Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye
In Brief Hall et al. profiled the proteome, transcriptome, and metabolome of the aging Drosophila eye. The integrated analysis revealed changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of immune response. - Research Article
Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis
In Brief We have characterized the cell-membrane N-glycome of two major developmental divisions of the brain, the forebrain and hindbrain, and three functional derivatives from them, including the cerebral cortex, hippocampus, and cerebellum, revealing an extraordinary diversity of N-glycans expressed in a global and functional region-specific manner in the adult mouse. Furthermore, we identified +25 N-glycans able to differentiate the forebrain and hindbrain N-glycome. Additionally, over 35 N-glycans distinguished the cortex, hippocampus, and cerebellum N-glycomes and may serve as region-specific glycan biomarkers. - Research Article
Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT
In Brief EMT can play a role in retinal diseases. Here, we present a comprehensive proteomic analysis aimed at defining the temporal protein expression changes associated with EMT of stem cell–derived retinal pigment epithelial cells. Tandem mass tag and direct data-independent acquisition MS approaches were performed after inducing RPE-EMT by enzymatic dissociation. We present integration of our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into the RPE-EMT progression. - Research Article
Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host–Pathogen Complexes
In Brief We present a pipeline that streamlines cross-linking mass spectrometry (XL-MS) data collection, data analysis, and integrative modeling of host–pathogen complexes. Using XL-MS, known atomic structures, and functional genetic data, we determined an integrative structure of the HIV-human A3G-CRL5-Vif-CBFβ complex. This structure illustrates HIV-1 Vif interaction with A3G and captures the structural dynamics and flexibility of the entire A3G-CRL5-Vif-CBFβ complex.
