Organellar Maps Through Proteomic Profiling – A Conceptual Guide

Borner, Georg H. H.

doi:10.1074/mcp.r120.001971

Cited by 38 publications

(56 citation statements)

References 59 publications

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“…To characterize the mechanism of antigen import enhancement, we developed a generic strategy to evaluate the biological activity of pharmacological compounds through comparative spatial proteomics ( Figure 2 A). Many, if not most, cell biological processes are accompanied by protein subcellular localization changes ( Lundberg and Borner, 2019 ; Borner, 2020 ). Hence, we adapted our previously developed method for generating organellar maps to pinpoint the subcellular localizations of thousands of proteins in a single experiment ( Itzhak et al., 2016 , 2017 , 2019 ).…”

Section: Resultsmentioning

confidence: 99%

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity

et al. 2020

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Summary Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.

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Section: Resultsmentioning

confidence: 99%

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity

et al. 2020

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“…Since marker proteins for different EV subtypes show overlapping profiles during density gradient centrifugation, proteomic analysis of peak fractions is insufficient to define EV compositions unambiguously. A more powerful approach is to profile the distribution of proteins across the entire gradient and to cluster proteins based on profile similarity (reviewed in (Borner, 2020)). However, the analysis of 12 fractions, across multiple replicates and biological conditions, requires considerable resources and is fraught with technical variability; the limited reproducibility of such experiments renders comparative applications challenging (Gatto et al, 2014).…”

Section: Unbiased Quantitative Proteomic Profiling Identifies Clustermentioning

confidence: 99%

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

et al. 2021

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Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.

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“…State-of-the-art high-throughput proteomics approaches are capable of identifying and detecting subtle changes in protein levels even for low abundance protein species [ 114 ], which make them an anticipated tool for EV research. Notably, the bulk of data generated to date has allowed the depiction of organelle maps by proteomic profiling, also covering the endosomal compartment (see [ 115 ] for a review). Far from classic label-free approaches, advances in relatively new labeling methods (i.e., TMT, iTRAQ, or SILAC) [ 116 ] and the application of robust quantification algorithms are also highly recommendable [ 117 ].…”

Section: Downstream Applications Using Ad-evsmentioning

confidence: 99%

Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells

Gómez-Serrano

Ponath

Preußer

et al. 2021

IJMS

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Adipose tissue and its crosstalk with other organs plays an essential role in the metabolic homeostasis of the entire body. Alteration of this communication (i.e., due to obesity) is related to the development of several comorbidities including type 2 diabetes, cardiovascular diseases, or cancer. Within the adipose depot, adipocytes are the main cell type and thus the main source of secreted molecules, which exert modulating effects not only at a local but also at a systemic level. Extracellular vesicles (EVs) have recently emerged as important mediators in cell–cell communication and account for part of the cellular secretome. In recent years, there has been a growing body of research on adipocyte-derived extracellular vesicles (Ad-EVs). However, there is still a lack of standardized methodological approaches, especially regarding primary adipocytes. In this review, we will provide an outline of crucial aspects when working on adipose-derived material, with a special focus on primary adipocytes. In parallel, we will point out current methodological challenges in the EV field and how they impact the transcriptomic, proteomic and functional evaluations of Ad-EVs.

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Organellar Maps Through Proteomic Profiling – A Conceptual Guide

Cited by 38 publications

References 59 publications

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity

Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells

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