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.

scholarly journals SAINT: probabilistic scoring of affinity purification–mass spectrometry data

2010 ◽  
Vol 8 (1) ◽  
pp. 70-73 ◽  
Author(s):  
Hyungwon Choi ◽  
Brett Larsen ◽  
Zhen-Yuan Lin ◽  
Ashton Breitkreutz ◽  
Dattatreya Mellacheruvu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Affinity Purification ◽  
Mass Spectrometry Data ◽  
Affinity Purification Mass Spectrometry

scholarly journals Mapping Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells

2021 ◽  
Author(s):  
Ankitha Shetty ◽  
Santosh D. Bhosale ◽  
Subhash Kumar Tripathi ◽  
Tanja Buchacher ◽  
Rahul Biradar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Differentiation ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Binding Partners ◽  
Th17 Cell Differentiation

Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell-differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among these, FOSL1 and FOSL2 influence the effector responses of Th17 cells. However, the molecular mechanisms underlying their functions are unclear, owing to the poorly characterized protein interaction networks of these factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification–mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a major fraction of these interactors to be associated with RNA binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17-fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL1 and FOSL2 that potentially govern Th17 cell-differentiation and associated pathologies.

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.

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