scholarly journals Epstein-Barr Virus LMP1 Modulates the CD63 Interactome

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 675
Author(s):  
Mujeeb Cheerathodi ◽  
Dingani Nkosi ◽  
Allaura S. Cone ◽  
Sara B. York ◽  
David G. Meckes
Keyword(s):  
Intracellular Signaling ◽  
Ribosomal Proteins ◽  
Protein Localization ◽  
Cell Communication ◽  
Enrichment Analysis ◽  
Surface Proteins ◽  
Rab Gtpases ◽  
Interacting Proteins ◽  
Sorting Nexins ◽  
Cargo Sorting

Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.

scholarly journals Epstein-Barr virus LMP1 modulates the CD63 interactome

2021 ◽  
Author(s):  
Mujeeb Cheerathodi ◽  
Dingani Nkosi ◽  
Allaura S. Cone ◽  
Sara B. York ◽  
David G. Meckes Jr.
Keyword(s):  
Intracellular Signaling ◽  
Ribosomal Proteins ◽  
Protein Localization ◽  
Cell Communication ◽  
Enrichment Analysis ◽  
Surface Proteins ◽  
Rab Gtpases ◽  
Interacting Proteins ◽  
Sorting Nexins ◽  
Cargo Sorting

Abstract Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains which associates with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in cellular trafficking of different proteins, EV cargo sorting and vesicles formation. We have preciously shown that CD63 is important in LMP1 trafficking to EVs and this also affects LMP1 mediated intracellular signaling including MAPK/ERK, NF-κB and mTOR activation. Using the BioID combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as proximal interacting newtwork of proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism and transportation. The CD63 only interactome was enriched in Rab GTPases, SNARE proteins and sorting nexins while adding LMP1 into the interactome increased presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of proteins enrichment from protein localization and vesicle mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTor, Nedd4L and PP2A indicating formation of a multiprotein complex with CD63 thereby potentially regulating LMP1 dependent mTor signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into network of partners required for endocytic trafficking, extracellular vesicle cargo sorting, formation and secretion.

scholarly journals Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Henning Großkopf ◽  
Sarah Vogel ◽  
Claudia Damaris Müller ◽  
Sebastian Köhling ◽  
Jan-Niklas Dürig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Protein Localization ◽  
Affinity Purification ◽  
Intracellular Localization ◽  
Enrichment Analysis ◽  
Transport Processes ◽  
Interacting Proteins ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Components

Abstract Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

The Corynebacterium diphtheriae HbpA hemoglobin-binding protein contains a domain that is critical for hemoprotein-binding, cellular localization and function.

2021 ◽  
Author(s):  
Lindsey R. Lyman ◽  
Eric D. Peng ◽  
Michael P. Schmitt
Keyword(s):  
Cellular Localization ◽  
Binding Protein ◽  
Protein Localization ◽  
Iron Acquisition ◽  
Corynebacterium Diphtheriae ◽  
Terminal Region ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Transport Systems ◽  
Iron Source

The acquisition of hemin-iron from hemoglobin-haptoglobin (Hb-Hp) by Corynebacterium diphtheriae requires the iron-regulated surface proteins HtaA, ChtA, ChtC, and the recently identified Hb-Hp binding protein HbpA. We previously showed that a purified form of HbpA (HbpA-S), lacking the C-terminal region, was able to bind Hb-Hp. In this study, we show that the C-terminal region of HbpA significantly enhances binding to Hb-Hp. A purified form of HbpA that includes the C-terminal domain (HbpA-FL) exhibits much stronger binding to Hb-Hp than HbpA-S. Size exclusion chromatography (SEC) showed that HbpA-FL as well as HtaA-FL, ChtA-FL, and ChtC-FL exist as high molecular weight complexes, while HbpA-S is present as a monomer, indicating that the C-terminal region is required for formation of large aggregates. Growth studies showed that expression of HbpA-FL in the Δ hbpA mutant restored wild-type levels of growth in low-iron medium that contained Hb-Hp as the sole iron source, while HbpA-S failed to complement the Δ hbpA mutant. Protein localization studies in C. diphtheriae showed that HbpA-FL is present in both in the supernatant and in the membrane fractions, and that the C-terminal region is required for membrane anchoring. Purified HbpA-FL was able to enhance growth of the Δ hbpA mutant when added to culture medium that contained Hb-Hp as a sole iron source, suggesting that secreted HbpA is involved in the use of hemin-iron from Hb-Hp. These studies extend our understanding of this novel Hb-Hp binding protein in this important human pathogen. IMPORTANCE Hemoproteins, such as Hb, are an abundant source of iron in humans and are proposed to be required by numerous pathogens to cause disease. In this report, we expand on our previous studies in further defining the role of HbpA in hemin-iron acquisition in C. diphtheriae . HbpA is unique to C. diphtheriae , and appears to function unlike any previously described bacterial iron-regulated Hb- or Hb-Hp-binding protein. HbpA is both secreted and present in the membrane, and exists as a large aggregate that enhances its ability to bind Hb-Hp and promote hemin-iron uptake. Current studies with HbpA will increase our understanding of iron transport systems in C. diphtheriae .

scholarly journals Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs

Blood ◽  
2020 ◽  
Vol 136 (20) ◽  
pp. 2346-2358 ◽  
Author(s):  
Özgün Babur ◽  
Alexander R. Melrose ◽  
Jennifer M. Cunliffe ◽  
John Klimek ◽  
Jiaqing Pang ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Intracellular Signaling ◽  
Vascular Inflammation ◽  
Functional Screening ◽  
Tandem Mass ◽  
Rab Gtpases ◽  
Platelet Surface ◽  
Form And Function ◽  
Signaling Mechanisms ◽  
And Function

Abstract Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

scholarly journals The GCP3-Interacting Proteins GIP1 and GIP2 Are Required for γ-Tubulin Complex Protein Localization, Spindle Integrity, and Chromosomal Stability

2012 ◽  
Vol 24 (3) ◽  
pp. 1171-1187 ◽  
Author(s):  
Natacha Janski ◽  
Kinda Masoud ◽  
Morgane Batzenschlager ◽  
Etienne Herzog ◽  
Jean-Luc Evrard ◽  
...  
Keyword(s):  
Protein Localization ◽  
Interacting Proteins ◽  
Chromosomal Stability ◽  
Complex Protein

p53 Dependent and Dose Dependent Effects of Rps29 Mutation In the Zebrafish Embryo.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1170-1170
Author(s):  
Alison M. Taylor ◽  
Jessica M. Humphries ◽  
Richard M. White ◽  
Ryan D. Murphey ◽  
Caroline E. Burns ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Board Of Directors ◽  
Ribosomal Proteins ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Ribosomal Protein Genes ◽  
Advisory Committees ◽  
Cardinal Vein ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Abstract 1170 Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by red cell aplasia and craniofacial abnormalities. Ribosomal protein genes are often mutated in patients with this disease, but the mechanism of action is still being investigated. To elucidate the effect of mutations in ribosomal proteins, we are studying a zebrafish rps29 mutant with hematopoietic and endothelial defects. Hematopoietic stem cells (HSCs) in rps29-/- embryos are significantly decreased, as assayed by runx1 and cmyb expression. Although the aorta and posterior cardinal vein form in the mutant, intersomitic vessel formation is affected. To test whether decreased p53 levels can rescue these defects, we crossed fish with mutated p53 into the rps29 background. In rps29-/-;p53-/- embryos, the vascular and HSC phenotypes are rescued, demonstrating that p53 may be required for these effects of rps29 knockdown. We performed a microarray comparing rps29-/- embryos and their siblings to identify genes that are differentially expressed in the mutant. Using gene set enrichment analysis (GSEA), we determined that the list of genes up-regulated in the rps29 mutant is enriched for genes up-regulated by p53 in response to irradiation. Many of the genes identified have known roles in apoptosis and stress response. We have also identified genes whose expression correlates with the number of wildtype copies of rps29. Orthopedia homolog a (otpa), which is specifically expressed in forebrain and hindbrain tissues at 24 hours post fertilization (hpf), is decreased in heterozygous siblings and further decreased in homozygous siblings. In addition, p53 knockdown partially increases otpa levels in the mutant. These data support a model where p53 activation is one of the critical downstream mediators of rps29 knockdown in several tissues, but the mechanism of tissue specificity remains unclear. The otpa phenotype suggests that regulation of some genes is dependent on rps29 levels. The zebrafish rps29 mutant will be a useful model for understanding how a decrease in ribosomal protein levels can cause specific defects in hematopoietic and neural tissues. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Lipid rafts sense and direct electric field-induced migration

2017 ◽  
Vol 114 (32) ◽  
pp. 8568-8573 ◽  
Author(s):  
Bo-jian Lin ◽  
Shun-hao Tsao ◽  
Alex Chen ◽  
Shu-Kai Hu ◽  
Ling Chao ◽  
...  
Keyword(s):  
Cell Migration ◽  
Membrane Proteins ◽  
Lipid Rafts ◽  
Electric Fields ◽  
Intracellular Signaling ◽  
Developmental Regulation ◽  
Primary Response ◽  
Surface Proteins ◽  
Dependent Manner ◽  
Frequency Dependent

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction.

scholarly journals Identification of Functional Interactome of Colistin Resistance Protein MCR-1 in Escherichia coli

2021 ◽  
Vol 11 ◽  
Author(s):  
Hui Li ◽  
Yingyu Wang ◽  
Qiyan Chen ◽  
Xi Xia ◽  
Jianzhong Shen ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Efflux Pump ◽  
Protein Biosynthesis ◽  
Rna Degradation ◽  
Interacting Proteins ◽  
Multidrug Efflux ◽  
Colistin Resistance ◽  
Multidrug Efflux Pump ◽  
Bait Protein ◽  
E Coli

The emergence and worldwide dissemination of plasmid-mediated colistin resistance gene mcr-1 has attracted global attention. The MCR-1 enzyme mediated colistin resistance by catalyzing phosphoethanolamine (PEA) transfer onto bacterial lipid A. However, the interaction partners of MCR-1 located in membrane protein in E. coli are unknown. Co-immunoprecipitation (Co-IP) and Mass Spectrometry were performed to define the interacting proteins of MCR-1. A total of three different anti-MCR-1 monoclonal antibody (mAbs) were prepared and 3G4 mAb was selected as the bait protein by compared their suitability for Co-IP. We identified 53, 13, and 14 interacting proteins in E. coli BL21 (DE3) (pET28a-mcr-1), E. coli BL21 (DE3) (pET28a-mcr-1-200), and E. coli DH5α (pUC19-mcr-1), respectively. Six proteins, including the stress response proteins DnaK (chaperone protein) and SspB (stringent starvation protein B), the transcriptional regulation protein H-NS, and ribosomal proteins (RpsE, RpsJ, and RpsP) were identified in all these three strains. These MCR-1-interacting proteins were mainly involved in ribosome and RNA degradation, suggesting that MCR-1 influences the protein biosynthesis through the interaction with ribosomal protein. Multidrug efflux pump AcrA and TolC were important interacting membrane proteins of MCR-1 referred to drug efflux during the PEA modification of the bacterial cell membrane. Overall, we firstly identified the functional interactome profile of MCR-1 in E. coli and discovered that two-component AcrA-TolC multidrug efflux pump was involved in mcr-1-mediated colistin resistance.

scholarly journals An Autoantigen Profile of Human A549 Lung Cells Reveals Viral and Host Etiologic Molecular Attributes of Autoimmunity in COVID-19

2021 ◽  
Author(s):  
Julia Y. Wang ◽  
Wei Zhang ◽  
Michael W. Roehrl ◽  
Victor B. Roehrl ◽  
Michael H. Roehrl
Keyword(s):  
Viral Infection ◽  
Ribosomal Proteins ◽  
Dermatan Sulfate ◽  
Protein Localization ◽  
Platelet Activating Factor ◽  
A549 Cells ◽  
Transcript Level ◽  
Smooth Muscle Contraction ◽  
Mrna Metabolism ◽  
Mitochondrial Ribosome

AbstractWe aim to establish a comprehensive COVID-19 autoantigen atlas in order to understand autoimmune diseases caused by SARS-CoV-2 infection. Based on the unique affinity between dermatan sulfate and autoantigens, we identified 348 proteins from human lung A549 cells, of which 198 are known targets of autoantibodies. Comparison with current COVID data identified 291 proteins that are altered at protein or transcript level in SARS-CoV-2 infection, with 191 being known autoantigens. These known and putative autoantigens are significantly associated with viral replication and trafficking processes, including gene expression, ribonucleoprotein biogenesis, mRNA metabolism, translation, vesicle and vesicle-mediated transport, and apoptosis. They are also associated with cytoskeleton, platelet degranulation, IL-12 signaling, and smooth muscle contraction. Host proteins that interact with and that are perturbed by viral proteins are a major source of autoantigens. Orf3 induces the largest number of protein alterations, Orf9 affects the mitochondrial ribosome, and they and E, M, N, and Nsp proteins affect protein localization to membrane, immune responses, and apoptosis. Phosphorylation and ubiquitination alterations by viral infection define major molecular changes in autoantigen origination. This study provides a large list of autoantigens as well as new targets for future investigation, e.g., UBA1, UCHL1, USP7, CDK11A, PRKDC, PLD3, PSAT1, RAB1A, SLC2A1, platelet activating factor acetylhydrolase, and mitochondrial ribosomal proteins. This study illustrates how viral infection can modify host cellular proteins extensively, yield diverse autoantigens, and trigger a myriad of autoimmune sequelae.

scholarly journals Integrin, Exosome and Kidney Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
An-Ran Shen ◽  
Xin Zhong ◽  
Tao-Tao Tang ◽  
Cui Wang ◽  
Jing Jing ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Intracellular Signaling ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Cell Communication ◽  
Transforming Growth Factor Β ◽  
Cellular Adhesion ◽  
Glomerular Sclerosis ◽  
Urokinase Plasminogen Activator Receptor ◽  
Focal Segmental Glomerular Sclerosis

Integrins are transmembrane receptors that function as noncovalent heterodimers that mediate cellular adhesion and migration, cell to cell communication, and intracellular signaling activation. In kidney, latency associated peptide-transforming growth factor β (TGF-β) and soluble urokinase plasminogen activator receptor (suPAR) were found as the novel ligands of integrins that contribute to renal interstitial fibrosis and focal segmental glomerular sclerosis glomerulosclerosis (FSGS). Interestingly, recent studies revealed that integrins are the compositional cargo of exosomes. Increasing evidence suggested that exosomal integrin played critical roles in diverse pathophysiologic conditions such as tumor metastasis, neurological disorders, immunology regulation, and other processes. This review will focus on the biology and function of exosomal integrin, emphasizing its potential role in kidney disease as well as its implications in developing novel therapeutic and diagnosis approaches for kidney disease.

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