International Journal of Molecular Sciences Article HIV-1 and Amyloid Beta Remodel Proteome of Brain Endothelial Extracellular Vesicles Ibolya E. András, Brice B. Sewell and Michal Toborek * Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33136-1019, USA; [email protected] (I.E.A.); [email protected] (B.B.S.) * Correspondence: [email protected] Received: 11 March 2020; Accepted: 7 April 2020; Published: 15 April 2020 Abstract: Amyloid beta (Aβ) depositions are more abundant in HIV-infected brains. The blood–brain barrier, with its backbone created by endothelial cells, is assumed to be a core player in Aβ homeostasis and may contribute to Aβ accumulation in the brain. Exposure to HIV increases shedding of extracellular vesicles (EVs) from human brain endothelial cells and alters EV-Aβ levels. EVs carrying various cargo molecules, including a complex set of proteins, can profoundly affect the biology of surrounding neurovascular unit cells. In the current study, we sought to examine how exposure to HIV, alone or together with Aβ, affects the surface and total proteomic landscape of brain endothelial EVs. By using this unbiased approach, we gained an unprecedented, high-resolution insight into these changes. Our data suggest that HIV and Aβ profoundly remodel the proteome of brain endothelial EVs, altering the pathway networks and functional interactions among proteins. These events may contribute to the EV-mediated amyloid pathology in the HIV-infected brain and may be relevant to HIV-1-associated neurocognitive disorders. Keywords: HIV-1; amyloid beta; extracellular vesicles; blood–brain barrier 1. Introduction HIV-infected brains tend to have enhanced amyloid beta (Aβ) deposition [1–6], mostly in the perivascular space [3,7–9]. Indeed, the blood–brain barrier (BBB) is thought to be a key player in the brain’s Aβ homeostasis [10]. It is now widely accepted that extracellular vesicles (EVs) may also be important in Aβ pathology [11–17]. Our earlier work has shown that HIV can increase the release of brain endothelial EVs and alter EV-Aβ levels. Moreover, brain endothelial cell-derived EVs can transfer Aβ to other cells of the neurovascular unit [18]. EVs carry specific cargo molecules, including a complex set of proteins, which can be transferred to the neighboring cells and affect their biology. Some of these proteins are on the EV surface. The surface proteins may allow for selective EV uptake by the recipient cells, like in the case of receptor-mediated endocytosis. Total proteomics can give detailed information on the EV protein cargo overall. Surface proteomics could indicate the “address” of a targeted delivery, while total proteomics would represent the delivered “package.” In this work, we investigated how exposure to HIV, alone and together with Aβ, impacts the surface and total proteomic landscape of EVs from human brain microvascular endothelial cells (HBMEC-EVs). By using this unbiased strategy, we obtained a complex, high-resolution insight into these changes. Int. J. Mol. Sci. 2020, 21, 2741; doi:10.3390/ijms21082741 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2741 2 of 39 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 39 2.2. ResultsResults 2.1.2.1. ExtracellularExtracellular VesiclesVesicles fromfrom HumanHuman BrainBrain MicrovascularMicrovascular EndothelialEndothelial CellsCells (HBMEC-EVs)(HBMEC-EVs) AreAre EnrichedEnriched withwith thethe MajorMajor EVEV MarkersMarkers AtAt first,first, wewe examinedexamined whetherwhether proteinsproteins thatthat areare frequentlyfrequently identifiedidentified inin EVsEVs/exosomes/exosomes fromfrom variousvarious sourcessources cancan bebe foundfound inin ourour isolatedisolated HBMEC-EVs.HBMEC-EVs. BasedBased onon thethe ExoCartaExoCarta EVEV proteomicsproteomics databasedatabase fromfrom didifferentfferent humanhuman cellcell typestypes thatthat havehave beenbeen isolatedisolated using didifferentfferent approachesapproaches [[19,2019,20],], wewe compiledcompiled thethe listlist ofof 100100 markermarker proteinsproteins thatthat areare mostmost oftenoften presentpresent onon EVsEVs (Table(Table1 ).1). The The surface surface HBMEC-EVHBMEC-EV proteome, which which contained contained a a total total of of 283 283 identified identified proteins, proteins, included included 62 62of the of thetop top100 100ExoCarta ExoCarta EV EVmarkers markers (Figure (Figure 1A,1A, Table Table 1).1). In addition,addition, thethe totaltotal HBMEC-EV HBMEC-EV proteome, proteome, which which containedcontained 501501 identifiedidentified proteins,proteins, includedincluded 8080 ofof suchsuch markersmarkers (Figure(Figure1 B,1B, Table Table1). 1). These These results results demonstratedemonstrate thatthat ourour HBMEC-EVHBMEC-EV isolationisolation waswas highlyhighly enrichedenriched withwith knownknown EV EV markers. markers. Figure 1. Cont. Int. J. Mol. Sci. 2020, 21, 2741 3 of 39 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 39 FigureFigure 1. Extracellular 1. Extracellular vesicle vesicle (EV)-specific (EV)-specific markersmarkers in in the the surface surface and and total total proteomes proteomes of human of human brain brain microvascularmicrovascular endothelial endothelial cells cells (HBMEC)-derived (HBMEC)-derived EVs. EVs. Venn Venn diagramdiagram showing the the overlap overlap between between the HBMEC-EVthe HBMEC surface-EV surface proteome proteome (283 proteins) (283 proteins) (A) or (A the) or HBMEC-EV the HBMEC total-EV total proteome proteome (501 (501 proteins) proteins) (B ) and the top(B) 100 and EV the marker top 100 proteins EV marker from proteins ExoCarta. from Cellular ExoCarta. component Cellular enrichment component ofenrichment the identified of the surface (C) andidentified total (D surface) EV proteomes. (C) and total The (D identified) EV proteomes. EV proteins The identified were enriched EV proteins for cellular were componentenriched for using the Scacellularffold component software. using the Scaffold software. 2.2. Cellular2.2. Cellular Component Component Enrichment Enrichmen oft of the the IdentifiedIdentified Surface and and Total Total EV EV Proteins Proteins UsingUsing the the Sca Scaffoldffold software, software, we we nextnext evaluatedevaluated the the HBMEC HBMEC-EV-EV proteins proteins according according to their to their knownknown cellular cellular localization. localization. This This approach approach may may indicate indicate the the parentparent cellularcellular compartment origin origin of of the the identified HBMEC-EV proteins. The majority of the HBMEC-EV surface proteins were identified HBMEC-EV proteins. The majority of the HBMEC-EV surface proteins were extracellular extracellular region proteins, followed by cytoplasmic, intracellular organelle, membrane, nuclear, regionendoplasmic proteins, followedreticulum, bycytoskeleton, cytoplasmic, Golgi, intracellular mitochondrial, organelle, endosomal, membrane, ribosomal nuclear,proteins, endoplasmicand one reticulum,unknown cytoskeleton, protein (Figure Golgi, 1C). mitochondrial, For the total HBMEC endosomal,-EV proteome, ribosomal the majority proteins, of andproteins one unknownwere proteincytoplasmic (Figure1 C).and For extracellular the total HBMEC-EVregion proteins proteome, (Figure 1D). the majority of proteins were cytoplasmic and extracellular region proteins (Figure 1D). 2.3. HIV and Aβ Exposure Results in Unique HBMEC-EV Proteome Signatures We next focused on the unique proteins induced by the exposure to HIV and Aβ. Comparison of the control vs. HIV surface HBMEC-EV proteomes identified 112 unique proteins in the control and Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 39 Int. J. Mol. Sci. 2020, 21, 2741 4 of 39 2.3. HIV and Aβ Exposure Results in Unique HBMEC-EV Proteome Signatures We next focused on the unique proteins induced by the exposure to HIV and Aβ. Comparison three unique proteins in the HIV group (Figure2A). By contrast, a similar comparison for the total of the control vs. HIV surface HBMEC-EV proteomes identified 112 unique proteins in the control proteome identified only three unique proteins in the control and as many as 259 unique proteins and three unique proteins in the HIV group (Figure 2A). By contrast, a similar comparison for the β intotal the proteome HIV group identified (Figure only2B). Comparisonthree unique ofproteins the surface in the proteomecontrol and between as many the as HIV259 unique vs. HIV +A groupsproteins identified in the HIV six group unique (Figure proteins 2B). in Comparison the HIV group of the and surface 116 unique proteome proteins between in thethe HIVHIV+ vsAβ. group (FigureHIV+Aβ2C). groups Finally, identified analysis six of unique the total proteins proteome in the revealed HIV group 28 unique and 116 proteins unique inproteins the HIV in groupthe and 201HIV+Aβ unique group proteins (Figure in 2C). the Finally, HIV+A analysisβ group of (Figure the total2D). proteome A list of revealed these unique 28 unique proteins proteins is in provided the in TablesHIV group2 and and3 for 201 the unique surface proteins and total in the proteomes, HIV+Aβ group respectively. (Figure 2D). A list of these unique proteins is provided in Table 2 and 3 for the surface and total proteomes, respectively. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 39 Figure 2. Cont. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 39 Int. J. Mol. Sci. 2020, 21, 2741 5 of 39 Figure 2. Cont. Int. J. Mol. Sci. 2020, 21, 2741 6 of 39 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW