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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130135	2/2	Homo sapiens	NAY	(d)(U)C DG	Lee JH	2013	33%
Study summary Full title HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases All authors Lee JH, Wittki S, Bräu T, Dreyer FS, Krätzel K, Dindorf J, Johnston IC, Gross S, Kremmer E, Zeidler R, Schlötzer-Schrehardt U, Lichtenheld M, Saksela K, Harrer T, Schuler G, Federico M, Baur AS Journal Mol Cell Abstract The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for rea (show more...)The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNF? converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNF? and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNF?. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD81/ ADAM17/ CD63/ CD9/ MHC1 non-EV: Proteomics no EV density (g/ml) 1.12-1.23 Show all info Study aim Other/Regulation of ADAM10 activation and secretion Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Wash: volume per pellet (ml) 35 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 110000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ MHC1/ ADAM17 ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ ADAM17 Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Wide-field Report size (nm) Not reported

	EV130135	1/2	Homo sapiens	Blood plasma	(d)(U)C	Lee JH	2013	11%
Study summary Full title HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases All authors Lee JH, Wittki S, Bräu T, Dreyer FS, Krätzel K, Dindorf J, Johnston IC, Gross S, Kremmer E, Zeidler R, Schlötzer-Schrehardt U, Lichtenheld M, Saksela K, Harrer T, Schuler G, Federico M, Baur AS Journal Mol Cell Abstract The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for rea (show more...)The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNF? converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNF? and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNF?. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment. (hide) EV-METRIC 11% (26th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: ADAM10/ TSG101/ ADAM17/ MHC1 non-EV: Proteomics no Show all info Study aim Other/Regulation of ADAM10 activation and secretion Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ MHC1/ ADAM10/ ADAM17 ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ ADAM10/ ADAM17 Characterization: Particle analysis None

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EV-TRACK ID	EV130135
species	Homo sapiens
sample type	Cell culture	Blood plasma
cell type	NAY	NA
condition	NAY	NAY
separation protocol	(d)(U)C DG	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	33	11