The World of Protein Interactions: Defining the Caveolin 3 Cardiac Interactome.

Abstract

HomeCirculation ResearchVol. 128, No. 6The World of Protein Interactions Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessEditorialPDF/EPUBThe World of Protein InteractionsDefining the Caveolin 3 Cardiac Interactome Jennifer E. Van Eyk Jennifer E. Van EykJennifer E. Van Eyk Correspondence to: Jennifer E. Van Eyk, PhD, Smidt Heart Institute, Cedars-Sinai Medical Center, Advanced Health Science Bldg, Room 370, 127 S San Vicente Blvd, Los Angeles, CA 90048. Email E-mail Address: [email protected] https://orcid.org/0000-0001-9050-148X Departments of Cardiology and Pathology, Smidt Heart Institute, Advanced Clinical BioSystems Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA. Search for more papers by this author Originally published18 Mar 2021https://doi.org/10.1161/CIRCRESAHA.121.318922Circulation Research. 2021;128:720–722This article is a commentary on the followingCaveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in CardiomyocytesArticle, see p e102Protein isoforms have highly similar amino acid sequences that arise from alternative splicing of the same gene or from the translation of different genes. Protein isoforms often perform similar but not necessarily identical biological roles, and in some cases, protein isoforms can assume entirely unique and distinct functions. A protein isoform can be constrained to a specific cell type or expressed only during a particular circumstance such as disease-induced perturbations or development stage. The underlying assumption in the case of cell-specific protein isoforms is that these are linked to a unique cell-specific function. In the case of CAV (caveolin) 3, its expression is predominantly constrained to straited muscle (heart and skeletal muscle), whereas other CAV isoforms are more widely expressed. In this study by Peper et al,1 the team uses a number of proteomic technologies to define the novel cell-specific interactome of CAV3 (compared with CAV1) and then proceed to define its unique functions within the cardiomyocyte.The CAV gene family (CAV1, CAV2, and CAV3) encodes 6 integral membrane isoforms, of which Cav 1 is known to have a splice variant, CAV2 has 2 splice variants, while CAV3 has no known splice variants. Caveolae are abundant complex plasma membrane pit-like invaginations that have roles in signaling, endocytosis, and can be mechanoresponsive. The CAVs are integral membrane proteins that dominate the membranes of caveolae where they act as scaffolds to provide order and compartmentalization of other protein components. They have similar protein structures, forms oligomers, and associate with cholesterol and sphingolipids to form the caveolae.Although CAV1 and CAV32–5 interactomes have previously been defined, they had not directly been identified in cardiac myocytes. Peper et al1 directly test the hypothesis that the isoform-specific interactome of CAV3, compared with that of CAV1, would define unique cardiomyocyte microdomains and functions. They used unbiased live-cell proximity proteomic (APEX) and isoform-specific affinity (classical immuno/affinity purification with and without cross-linking) in cardiac myocytes, to show that the monocarboxylate transporter McT1 (monocarboxylate transporter 1) (and transferrin receptor protein 1 [TfR1]) specifically binds CAV3 while aquaporin1 binds uniquely to CAV1. The authors went on to use super-resolution STED (Stimulated Emission Depletion) microscopy to show that these unique CAV1 and CAV3 interactomes result in distinct distributions within cardiomyocyte transverse tubules. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9)-mediated CAV3 knockout, they additionally established a stabilizing role for McT1 surface expression and directly showed that the CAV3 interactome has a role in proton-coupled lactate shuttling, late sodium currents, and in the early afterdepolarizations at least, in human iPSC-derived cardiomyocytes. Finally, this team defined that within the cardiac myocyte, there resides a core complex consisting of CAV3, McT1, Ncx1 (sodium/calcium exchanger), and Na/K-ATPase. These novel insights are exciting and may someday serve as important targets for the treatment of human reentry arrhythmias.The study by Peper et al showcases the application of proteomic tools and technologies to yield novel biologically important insights. The study also highlights the power and need for subsequent downstream experiments, to link a protein discovery with its functional relevance. The field of proteomics is not a single approach but rather an array of technologies and methodologies, often converging on mass spectrometry to provide the analytical readout. The evergrowing suite of proteomic technologies is increasingly able to capture the chemical and biological diversity of the proteome: where protein regulation can occur via changes in concentration or through chemical alterations to the amino acid sequence due differential isoform expression or co- and posttranslational modifications. In the case of the two CAV3 isoforms in comparison with CAV1, the binding partners and protein complexes differ, resulting in distinct cell location (and presumably structures), as well as function(s) within the heart.Proteins live in an interwoven community, and the quantification of a protein’s interacting partners within a cell has led to the growing appreciation that the cellular location of proteins and their complexes is highly regulated, as are the movements and translocations of these entities throughout the cell. Spatial proteomics—a collection of approaches with their roots in classical protein biochemistry techniques like immunohistochemistry—has evolved to include measurements that define the protein interactome, describe protein neighborhoods, and elucidate protein structure.6 Proximity-dependent biotinylation (PDB) approaches (eg, APEX and BioID) are able to identify the proteins located near their targets in a living cell.7 APEX is based on peroxidases while biotin protein ligases are used with BioID. There has been continuous innovation in this field, from new reagents to strategies for PDB, such as split PDB where the APEX molecule is split into 2 fragments that are each inactive until located in close proximity. The strength of the PDB approach lays in the precision with which it can provide quantitative data within a living cell. Conversely, the challenge is protein communities are identified and not necessarily only the protein complex nor its direct primary binding partners. The use of unnatural amino acids8 as cross-linker is an elegant approach allowing one to specifically identify primary binding partners and define the binding interface under conditions as similar as possible to those encountered by the native protein. The use of photo-activated unnatural amino acid cross-linkers that contain aryl azide, benzophenone, and diazirines, generates reactive species by irradiating live cells with UV light.9 Mass spectrometry, as with the PDB approaches, is used to identify the protein complex, and in the case of unnatural amino acid cross-linking, the actual amino acid residues in proximity to the binding site (if not the binding site itself) are revealed. The identification of whole cell protein complexes, 100s of protein complexes, and the ability to quantify their changes in a living cell is quickly emerging.10The biology that is captured by the interactome and spatial proteomics is of great interest, especially when it is altered by a physiological or pathological perturbation. Being able to define whether a protein is modified, or assumes a specific isoform, is also within the domain of proteomics. Structural analysis of intact proteins for isoforms, along with co- and posttranslational modifications, is rapidly being advanced by the growth and development of top-down mass spectrometry11 and encompasses studies where cardiac specific isoforms are being defined.12 Analysis of mass spectrometry data to help stitch together the unique sequences that define isoforms, be it from different genes or from splice variants, can get complicated very quickly as some splice variants arise with >1 difference. The development of software13 in this space is an ongoing challenge. The exact composition of any proteome is unknown14,15 due to dynamics and also technology but defining the interactomes of proteoforms and directly elucidating function is key, as elegantly demonstrated by Peper et al for CAV1 compared with CAV3. Proteomics is geared to capture the immense landscape of the proteome complexity using quantitative and analytical robust methods.AcknowledgmentsWe thank Drs Koen Raedschelders and John R. Yates, III for knowledge and insight.Sources of FundingFunding support was provided by R01HL111362 WFUHS117899 and R01HL14450.Disclosures None.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.For Sources of Funding and Disclosures, see page 721.Correspondence to: Jennifer E. Van Eyk, PhD, Smidt Heart Institute, Cedars-Sinai Medical Center, Advanced Health Science Bldg, Room 370, 127 S San Vicente Blvd, Los Angeles, CA 90048. Email jennifer.[email protected]org