Quantitative Proteomics of Caveolin-1-regulated Proteins

Abstract

Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. Molecular & Cellular Proteomics 9:2109 -2124, 2010.Caveolae have been extensively studied and shown to play an important role in the regulation of many cellular functions including cell signaling, vesicular transport, and lipid metabolism. Caveolae are especially abundant in adipocytes, endothelial cells (ECs), 1 fibroblasts, and smooth muscle cells (1-3), and there is evidence that the loss of caveola function may contribute to dyslipidemia, muscular dystrophy, cancer, diabetes, and cardiovascular diseases (1, 4 -7). Caveolae are a subset of lipid rafts characterized by a particularly high content of cholesterol and sphingolipids that contain the protein caveolin. There are three isoforms of caveolin, caveolin-1 (Cav-1), Cav-2, and Cav-3, that have been extensively studied (1, 4). Cav-1 and Cav-3 are required for caveola formation in different tissues (8 -10). Although Cav-2 is not required for caveola formation per se, (11) it regulates aspects of lung morphogenesis. Although the roles of caveolins in caveola formation are well known, the requirement of other proteins regulating caveolin function and thus caveola formation has been recently addressed (12)(13)(14).To identify proteins that stably interact and are regulated by Cav-1, we used an unbiased proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ). iTRAQ is a powerful quantitative proteomics tool for the relative quantification of proteins in complex mixtures by mass spectrometry (15), and this technology has been successfully used for global comparison of protein expression in many cells systems and in different tissues (16,17). As caveolae and Cav-1 ar...

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos

“…Justificando a maior expressão da caveolina -1 em relação à caveolina -2, Deurs et al (2003), Dávalos et al (2010), Hansen & Nichols (2010) afirmam que a expressão das caveolina -1, a principal proteína de revestimento da cavéola, é necessária para a estruturação da cavéola, em células não musculares, já a caveolina -3 faz parte da biogênese da cavéola presente na musculatura cardíaca e esquelética à medida que somente a presença da caveolina -2 não induz a sua formação. Ainda é fundamental a presença da proteína caveolina -1 na cavéola para que a proteína caveolina -2 seja enviada do complexo de Golgi para a mesma, caso contrário a caveolina -2 seria degradada no próprio complexo de Golgi (Okamoto et al 1998, Razani et al 2001.…”

“…Dávalos et al (2010) completou dizendo que as cavéolas são micro estruturas encontradas de forma abundante na membrana plasmática de muitas cé-lulas especializadas, incluindo células endoteliais, células epiteliais, e adipócitos. Com a microscopia atual, pode-se notar que há variedade morfológica destas estruturas de membranas, podendo assim estar dispostas em grupos denominados "rosetas" e/ou "cachos de uvas.…”

Hemoglobin α in the blood vessel wall

“…Today, this paradigm in RBC remains unchanged. However we 3 , and others 4,5 , have demonstrated that Hbα is expressed in the blood vessel wall but in contrast to other globins in the blood vessel wall 3,6,7 , Hbα localization and physiological effects are concentrated at myoendothelial junctions (MEJs) in the endothelial cells (EC) lining the lumen of blood vessels.…”

Uncoupling Caveolae From Intracellular Signaling In Vivo

“…Previous studies have shown that the genetic loss of Cav-1 destabilizes caveolin-2 (Cav-2) and to a lesser extent destabilizes the caveolin adaptor, Cavin-1 7, 9, 29 . Interestingly, Cav-1-F92A expression in EC enhances Cav-2 and Cavin-1 levels in whole lung lysates from transgenic mice (Figure 3A) demonstrating that Cav-1-F92A does not impair caveolae integrity, but likely stabilizes the protein complex.…”

“…Despite these findings, it is well known, that Cav-1 KO animals exhibit both pulmonary and cardiac fibrosis 7, 30, 31 and altered signaling 7 . A robust readout of caveolae formation and function is measuring the Cav-2 protein level and to a lower extent Cavin-1, as both proteins are in a stable protein complex with Cav-1, and the loss of Cav-1 genetically destabilizes endogenous Cav-2 and Cavin-1 7, 9, 29 . Whole lung or cardiac lysates show that EC expression of Cav-1-F92A indeed increased Cav-2 protein levels, suggesting that the point mutant F92A isoform did not disrupt the integrity of caveolae.…”