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Caveolins are lipid-binding proteins that can organize membrane remodeling and oligomerize into the 8S complex. The CAV1-8S complex comprises a disk-like structure, about 15 nm in diameter, with a central beta barrel. Further oligomerization of 8S complexes remodels the membrane into caveolae vessels, with a dependence on cholesterol concentration. However, the molecular mechanisms behind membrane remodeling and cholesterol filtering are still not understood. Performing atomistic molecular dynamics simulations in combination with advanced sampling techniques, we describe how the CAV1-8S complex bends the membrane and accumulates cholesterol. Here, our simulations show an enhancing effect by the palmitoylations of CAV1, and we predict that the CAV1-8S complex can extract cholesterol molecules from the lipid bilayer and accommodate them in its beta barrel. Through backmapping to the all-atom level, we also conclude that the Martini v.2 coarse-grained force field overestimates membrane bending, as the atomistic simulations exhibit only very localized bending., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Caveolin-1 (Cav1) is a 22 kDa intracellular protein that is the main protein constituent of bulb-shaped membrane invaginations known as caveolae. Cav1 can be also found in functional non-caveolar structures at the plasma membrane called scaffolds. Scaffolds were originally described as SDS-resistant oligomers composed of 10-15 Cav1 monomers observable as 8S complexes by sucrose velocity gradient centrifugation. Recently, cryoelectron microscopy (cryoEM) and super-resolution microscopy have shown that 8S complexes are interlocking structures composed of 11 Cav1 monomers each, which further assemble modularly to form higher-order scaffolds and caveolae. In addition, Cav1 can act as a critical signaling regulator capable of direct interactions with multiple client proteins, in particular, the endothelial nitric oxide (NO) synthase (eNOS), a role believed by many to be attributable to the highly conserved and versatile scaffolding domain (CSD). However, as the CSD is a hydrophobic domain located by cryoEM to the periphery of the 8S complex, it is predicted to be enmeshed in membrane lipids. This has led some to challenge its ability to interact directly with client proteins and argue that it impacts signaling only indirectly via local alteration of membrane lipids. Here, based on recent advances in our understanding of higher-order Cav1 structure formation, we discuss how the Cav1 CSD may function through both lipid and protein interaction and propose an alternate view in which structural modifications to Cav1 oligomers may impact exposure of the CSD to cytoplasmic client proteins, such as eNOS., (© 2024 The Author(s).)

Caveolin-1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin-1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within the C-terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full-length S-alkylated caveolin-1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI-LC-MS analyses verified the addition of the C16 alkyl groups to caveolin-1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed that S-alkylation does not significantly affect the global helicity of the protein; however, molecular dynamics simulations revealed that there were local regions where the helicity was altered positively or negatively by S-alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of the C-terminal domain, resulting in a disposition within the bilayer characterized by increased depth., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P 2 ]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Diabetes mellitus (DM) can be implicated in the perturbations of vascular integrity and the dysfunction of angiogenesis. Chitosan has the advantage of promoting the vascular endothelial cell proliferation. However, the molecular mechanism of action in the promotion of wound healing by chitosan derivatives is still debated. In the current study, DM with chronic wound (CW) model rats were prepared and treated with chitosan. Vascular endothelial cells isolated from granulation tissues were conducted by RNA sequencing. Two thousand three hundred and sixteen genes were up-regulated, while 1,864 genes were down-regulated after chitosan treatment compared to CW group. Here, we observed that caveolin 1 (CAV1) was highly expressed induced by chitosan. Furthermore, we observed that CAV1 knockdown could compromise the activation of Wnt pathway by reduction of β-catenin in rat aortic endothelial cells (RAOECs) and brain endothelium four cells (RBE4s). Moreover, we determined a direct interaction between CAV1 and β-catenin by IP assay. The C-terminus of CAV1 and β-catenin (24 to 586 amino acids) contributed to the interaction of these two proteins. Finally, the protein docking analysis indicated that the fragments of β-catenin (253-261 'FYAITTLHN' and 292-303 'KFLAITTDCLQI') might have affected the structure by CAV1 and facilitated the resistance to degradation. Taken together, our study demonstrates that chitosan can up-regulate CAV1 expression, and CAV1 can interact with β-catenin for promotion of canonical Wnt signaling pathway activity. Our results deepens the molecular mechanism of the Wnt pathway in vascular endothelial cells and is beneficial to developing new targets to assist in enhancing the pharmacological effect of chitosan on wound healing and angiogenesis against DM.

Background: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown., Methods: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START., Results: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties., Conclusion: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins., (© 2021. The Author(s).)

Caveolin-1 (CAV1), a membrane protein that is necessary for the formation and maintenance of caveolae, is a promising drug target for the therapy of various diseases, such as cancer, diabetes, and liver fibrosis. The biology and pathology of caveolae have been widely investigated; however, very little information about the structural features of full-length CAV1 is available, as well as its biophysical role in reshaping the cellular membrane. Here, we established a method, with high reliability and reproducibility, for the expression and purification of CAV1. Amyloid-like properties of CAV1 and its C -terminal peptide CAV1(168-178) suggest a structural basis for the short linear CAV1 assemblies that have been recently observed in caveolin polyhedral cages in Escherichia coli (E. coli). Reconstitution of CAV1 into artificial lipid membranes induces a caveolae-like membrane curvature. Structural characterization of CAV1 in the membrane by solid-state nuclear magnetic resonance (ssNMR) indicate that it is largely α-helical, with very little β-sheet content. Its scaffolding domain adopts a α-helical structure as identified by chemical shift analysis of threonine (Thr). Taken together, an in vitro model was developed for the CAV1 structural study, which will further provide meaningful evidences for the design and screening of bioactive compounds targeting CAV1.

The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82-101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1-101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis., (© 2021 Wiley-VCH GmbH.)

Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.

Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here, we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin-1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodeling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking, and signaling., (© 2021 Zhou et al.)

The plasma membrane is a dynamic lipid bilayer that engages with the extracellular microenvironment and intracellular cytoskeleton. Caveolae are distinct plasma membrane invaginations lined by integral membrane proteins Caveolin1, 2, and 3. Caveolae formation and stability is further supported by additional proteins including Cavin1, EHD2, Pacsin2 and ROR1. The lipid composition of caveolar membranes, rich in cholesterol and phosphatidylserine, actively contributes to caveolae formation and function. Post-translational modifications of Cav1, including its phosphorylation of the tyrosine-14 residue (pY14Cav1) are vital to its function in and out of caveolae. Cells that experience significant mechanical stress are seen to have abundant caveolae. They play a vital role in regulating cellular signaling and endocytosis, which could further affect the abundance and distribution of caveolae at the PM, contributing to sensing and/or buffering mechanical stress. Changes in membrane tension in cells responding to multiple mechanical stimuli affects the organization and function of caveolae. These mechanical cues regulate pY14Cav1 levels and function in caveolae and focal adhesions. This review, along with looking at the mechanosensitive nature of caveolae, focuses on the role of pY14Cav1 in regulating cellular mechanotransduction.

Caveolae are defined as 50-100 nm wide pits in the plasma membrane containing oligomeric caveolin proteins. They have been implicated in endocytosis (including phagocytosis), transcytosis, calcium signalling, and numerous other signal transduction events. Caveolin-1, a major structural component of caveolae, enhances Rab5 activity. In this study, we examined the effect of a synthetic cell-permeable peptide of the caveolin-1 scaffolding domain (CSD) on phagocytosis. Treatment with the CSD peptide increased Rab5 activity, Rab5-early endosome antigen 1 (EEA1) interaction, and phagocytosis of Escherichia coli. The results suggest that the synthetic cell-permeable CSD peptide is an activator of phagocytosis.

Caveolae consist in lipid raft domains composed of caveolin proteins, cholesterol, glycosphingolipids, and GPI-anchored proteins. Caveolin proteins present three different types, caveolin 1 (CAV-1), caveolin 2 (CAV-2) and caveolin 3 (CAV-3), with a very similar structure and amino acid composition. The native caveolin proteins oxidation mechanism was investigated for the first time, at a glassy carbon electrode, using cyclic, square wave and differential pulse voltammetry. The three native caveolin proteins oxidation mechanism presented only one tyrosine and tryptophan amino acid residues oxidation peak. Denatured caveolin proteins presented also the tyrosine, tryptophan and cysteine amino acid residues oxidation peaks. The reverse cholesterol transport is related to caveolae and caveolin proteins, and CAV-1 is directly connected to cholesterol transport. The influence of cholesterol on the three caveolin proteins electrochemical behaviour was evaluated. In the absence and in the presence of cholesterol, significant differences in the CAV-1 oxidation peak current were observed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A significant hurdle in obtaining biophysical information on membrane proteins is developing a successful strategy for their reconstitution into a suitable membrane mimic. In particular, utilization of the more 'native-like' membrane mimics such as bicelles is generally more challenging than simple micellar solubilization. Caveolin-1, an integral membrane protein involved in membrane curvature, endocytosis, mechano-protection, and signal transduction, has been shown to be particularly recalcitrant to standard reconstitution protocols due to its highly hydrophobic characteristics. Herein we describe a robust method to incorporate recombinantly produced full-length caveolin-1 into bicelles at levels needed for biophysical experimentation. The benchmark of successful reconstitution is the obtainment of protein in a homogeneous state; therefore, we developed a validation procedure to monitor the success of the reconstitution using analytical ultracentrifugation of density-matched bicelles. Our findings indicated that our protocol produces a very homogeneous preparation of caveolin-1 associated with bicelles, and that caveolin-1 is highly α-helical (by circular dichroism spectroscopy). We believe that this methodology will serve as a general strategy to facilitate biophysical studies on membrane proteins., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-β1 (Ad-TGF-β1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Emerging evidence suggests that extracellular vesicle (EV)-containing miRNAs mediate intercellular communications in response to noxious stimuli. It remains unclear how a cell selectively sorts the cellular miRNAs into EVs. We report that caveolin-1 (cav-1) is essential for sorting of selected miRNAs into microvesicles (MVs), a main type of EVs generated by outward budding of the plasma membrane. We found that cav-1 tyrosine 14 (Y14)-phosphorylation leads to interactions between cav-1 and hnRNPA2B1, an RNA-binding protein. The cav-1/hnRNPA2B1 complex subsequently traffics together into MVs. Oxidative stress induces O -GlcNAcylation of hnRNPA2B1, resulting in a robustly altered hnRNPA2B1-bound miRNA repertoire. Notably, cav-1 pY14 also promotes hnRNPA2B1 O -GlcNAcylation. Functionally, macrophages serve as the principal recipient of epithelial MVs in the lung. MV-containing cav-1/hnRNPA2B1 complex-bound miR-17/93 activate tissue macrophages. Collectively, cav-1 is the first identified membranous protein that directly guides RNA-binding protein into EVs. Our work delineates a novel mechanism by which oxidative stress compels epithelial cells to package and secrete specific miRNAs and elicits an innate immune response., (© 2019 Lee et al.)

The structure and dynamics of a truncated (residues 82-136) caveolin-1 (Cav1) construct having a helix-break-helix motif are explored by both all-atom free energy and molecular dynamics (MD) simulations in an explicit bilayer membrane. Two stable Cav1 conformations with small (LB-Cav1) and large hinge angles (RB-Cav1) between two helices are identified although their relative free energy cannot be reliably estimated due to the sampling issues. RB-Cav1s contain one or two lipids residing between the helices that are hydrogen bonded (h-bonded) to both helices in a multidentate fashion. LB-Cav1s show the helices with mono-dentate lipid h-bond interactions or multidentate interactions limited to a single helix at most. The two conformational states of Cav1 remain their initial state during 2-μs MD simulation, suggesting that there is a significant hidden barrier (other than the insertion depth of Cav1 and its hinge angle) and the Cav1 conformational states are tightly regulated by the h-bonds between Cav1 and lipids along with the associated lipid rearrangement during the course of Cav1 conformational changes. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Caveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT-3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.

Atherosclerosis is a lipid disease characterized by accumulation of low density lipoprotein (LDL) in the artery wall. The transport of LDL across the endothelium of coronary artery is an initiating event of atherosclerosis, whose mechanism remains poorly understood. In the last decade, it has been shown that in caveolin-1 (Cav-1) deficient mice, LDL infiltration in aorta wall is decreased and CD36 expression in aortas is down-regulated, leading to regression of atherosclerotic lesions. In the present study, we show that native LDL endocytosis is decreased in endothelial cells deficient in Cav-1 or CD36. We demonstrate that Cav-1 and CD36 interact in caveolae-rich domains by different biochemical approaches. In addition, confocal microscopy reveals some colocalization of Cav-1 with CD36. These findings indicate that caveolae and CD36 are involved in native LDL endocytosis and suggest that CD36 might be a good candidate for the transport of native LDL across the endothelium, an early event in atherosclerosis., (Copyright © 2019. Published by Elsevier B.V.)

Cancer stem cells (CSCs) are responsible for tumor initiation, metastasis and recurrence. Caveolin-1 (Cav-1) is a major protein of caveolae, which participates in various cellular functions, such as vesicle trafficking, cholesterol homeostasis, tumor progression, etc. In the present study, we investigated a role for Cav-1 in regulating the stemness of human breast cancer (MDA-MB-231) cells. Cav-1 expression was significantly lower in tumorspheres than in adherent cells. The silencing of Cav-1 enhanced stemness of MDA-MB-231 cells. Mechanistically, Cav-1 silencing was accompanied by enhanced expression of Bmi-1, which is a representative self-renewal regulator, and promoted epithelial-mesenchymal transition. In a CSC-like state, reduced Cav-1 depends on its destabilization through ubiquitin-proteasome degradation. We further found that Src-mediated phosphorylation of Cav-1 at the Tyr 14 residue is essential for its degradation. Taken together, these findings suggest that Cav-1 destabilization by Src may play a pivotal role in manifestation and maintenance of stemness in breast cancer cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Caveolin-1 (cav-1) is an important player in cell signaling and endocytosis that has been shown to colocalize with cholesterol-rich membrane domains. Experimental studies with varying cav-1 constructs have suggested that it can induce both cholesterol clustering and membrane curvature. Here, we probe the molecular origin of membrane curvature and cholesterol clustering by cav-1 by using coarse-grain molecular dynamics simulations. We have performed a series of simulations of a functionally important cav-1 construct, comprising the membrane-interacting domains and a C-terminal palmitoyl tail. Our results suggest that cav-1 is able to induce cholesterol clustering in the membrane leaflet to which it is bound as well as the opposing leaflet. A positive membrane curvature is observed upon cav-1 binding in cholesterol-containing bilayers. Interestingly, we observe an interplay between cholesterol clustering and membrane curvature such that cav-1 is able to induce higher membrane curvature in cholesterol-rich membranes. The role of the cav-1 palmitoyl tail is less clear and appears to increase the membrane contacts. Further, we address the importance of the secondary structure of cav-1 domains and show that it could play an important role in membrane curvature and cholesterol clustering. Our work is an important step toward a molecular picture of caveolae and vesicular endocytosis., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1; possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo.

Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cav sol , for expression in E. coli. Cav sol folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and the polymerase I and transcript release factor. As expected, cav sol does not bind off-target proteins. Cellular studies show that cav sol retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cav sol forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cav sol 's secondary structure is a mixture of α-helices and β-strands. Isothermal titration calorimetry experiments reveal that cav sol binds to gp41 and PKA with low micromolar binding affinity (K D ). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have previously reported that the CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, elicits peptide-specific antibodies. Here, we have investigated the cellular immune response and the protective efficacy against a simian/human immunodeficiency virus (SHIV 162P3 ) challenge. In addition to the CBD1 peptide, peptides overlapping the caveolin-binding-motif (CBM) ( 622 IWNNMTWMQW 631 or 622 IWNNMTW 628 ) were fused to a Gag-p24 T helper epitope for vaccination. All immunized cynomolgus macaques responded to a cocktail peptide immunization by inducing specific T cells and the production of high-titer CBD1/CBM peptide-specific antibodies. Six months after the fourth vaccine boost, six control and five vaccinated animals were challenged weekly by repeated exposure to SHIV 162P3 via the mucosal rectal route. All control animals were infected after 1 to 3 challenges with SHIV, while among the five vaccinated monkeys, three became infected after a delay compared to control; one was infected after the eighth viral challenge, and one remained uninfected even after the ninth SHIV challenge. Immunized animals maintained a CD4 T cell count, and their central memory CD4 T cells were less depleted than in the control group. Furthermore, SHIV challenge stimulates antigen-specific memory T cell response in vaccinated macaques. Our results indicate that peptides derived from the CBM region can be immunogenic and provide protection against SHIV infection in cynomolgus monkeys. IMPORTANCE In HIV-1-producing cells, gp41 exists in a complexed form with caveolin-1, an interaction most probably mediated by the caveolin-1 binding motif. This sequence is highly conserved in every single HIV-1 isolate, thus suggesting that there is constant selective pressure to preserve this sequence for a specific function in the HIV infectious cycle. Consequently, the CBM sequence may represent the "Achilles' heel" of HIV-1 in the development of an efficient vaccine. Our results demonstrate that macaques immunized with the CBM-based peptides displayed a delay in the onset of viral infection and CD4 depletion, as well as a significant induction of antigen-specific memory T cell response, which is essential for the control of HIV/SIV infections. Finally, as HIV-infected individuals lack anti-CBM immune responses, CBM-based vaccines could have applications as a therapeutic vaccine in AIDS patients., (Copyright © 2018 American Society for Microbiology.)

The process by which muscle fiber electrical depolarization is linked to activation of muscle contraction is known as excitation-contraction coupling (ECC). Our understanding of ECC has increased enormously since the early scientific descriptions of the phenomenon of electrical activation of muscle contraction by Galvani that date back to the end of the eighteenth century. Major advances in electrical and optical measurements, including muscle fiber voltage clamp to reveal membrane electrical properties, in conjunction with the development of electron microscopy to unveil structural details provided an elegant view of ECC in skeletal muscle during the last century. This surge of knowledge on structural and biophysical aspects of the skeletal muscle was followed by breakthroughs in biochemistry and molecular biology, which allowed for the isolation, purification, and DNA sequencing of the muscle fiber membrane calcium channel/transverse tubule (TT) membrane voltage sensor (Cav1.1) for ECC and of the muscle ryanodine receptor/sarcoplasmic reticulum Ca 2+ release channel (RyR1), two essential players of ECC in skeletal muscle. In regard to the process of voltage sensing for controlling calcium release, numerous studies support the concept that the TT Cav1.1 channel is the voltage sensor for ECC, as well as also being a Ca 2+ channel in the TT membrane. In this review, we present early and recent findings that support and define the role of Cav1.1 as a voltage sensor for ECC.

Liver fibrosis is the common pathological process characterized by activation of hepatic stellate cells (HSCs) and overproduction of extracellular matrix (ECM). Caveolin-1 (Cav1), the principal component of caveolae, is regarded as an important inhibitor of multiple signaling molecules including transforming growth factor β1(TGF-β1) signaling. To evaluate the role of Cav1 in liver fibrosis, Cav1 deficient (Cav1 −/− ) and wild type (WT) mice were subjected to liver fibrosis induced by carbon tetrachloride (CCl₄). Results indicated no significant difference between Cav1 −/− and WT mice in inflammation or collagen content before CCl₄ treatment. After CCl₄ administration, Cav1 −/− mice showed enhanced TGF-β1 signaling, as reflected by a significantly greater amount of phosphorylation of Smad2 and collagen deposition in livers over WT animals. Qualitative and quantitative analysis indicated that inflammatory injury to the liver was markedly aggravated, accompanied by increased degeneration and necrosis of hepatocytes, higher alanine aminotransferase (ALT)/aspartate aminotransferase (AST), TGF-α and IL-1β levels in Cav1 −/− animals. The mRNA and protein levels of α-smooth muscle actin (α-SMA), Collagen α1(I), and Collagen α1(III) were further enhanced in Cav1 −/− animals. We also observed a significant decrease in collagen content in Cav1 −/− and WT animals administrated with Cav1 scaffolding domain peptides (CSD). In vitro study indicated that phosphorylation of Smad2 was inhibited after CSD treatment, accompanied by decreased protein levels of α-SMA, Collagen α1(I), and Collagen α1(III) in HSCs. We conclude that Cav1 is an important inhibitor of TGF-β1/Smad signaling in HSCs activation and collagen production, which might make it a promising target for therapy of liver fibrosis.

Aim: To track early events during lung metastasis, we labeled cells expressing (B16F10 CAV1 ) or lacking CAV1 (B16F10 mock ) with gold nanoparticles conjugated to the peptide TAT (AuNPs-PEG-TAT)., Methods: B16F10 expressing or lacking CAV1 were labeled with AuNPs-PEG-TAT. The physicochemical properties and cytotoxicity of these nanoparticles, as well as their effects on migration and invasiveness of B16F10 cells in vitro were evaluated. Ex vivo lung distribution of the labeled cells after tail vein injection into C57BL/6 mice was examined., Results: AuNPs-PEG-TAT did not affect B16F10 viability, migration and invasiveness. The metastatic and tumorigenic capability of the labeled B16F10 was also not modified in comparison to unlabeled B16F10 cells. CAV1 expression favored the retention of B16F10 cells in the lungs of mice 2 h post injection, suggesting CAV1 promoted adherence to endothelial cells and transendothelial migration., Conclusions: We developed a protocol to label B16F10 cells with AuNPs-PEG-TAT that permits subsequent tracking of cells in mice. CAV1 overexpression was found to increase retention and transendothelial migration of B16F10 cells in the lung.

Aim: To develop a nanocarrier for targeted delivery of agents to the cartilage., Materials & Methods: Chondrocyte affinity peptide modified PEGylated polyamidoamine conjugates (CAP-PEG-PAMAM) were prepared and rhodamine B isothiocyanate (RB) fluorophore was linked on them for comparative biological tracing and profiling., Results: CAP4-PP-RB exhibited much more efficient cellular uptake in vitro than that of PEG-PAMAM-RB. Both the conjugates were likely internalized by chondrocytes via clathrin and caveolin co-mediated endocytosis, and delivered to lysosomes. In vivo imaging demonstrated the fluorescein-labeled nanocarrier was capable to persist in the joint cavity of rats for a prolonged time. Furthermore, the CAP4-PEG-PAMAM showed a good biocompatibility and enhanced penetration effects in vivo., Conclusion: CAP-PEG-PAMAM could be an effective nanocarrier for intra-articular delivery of agents to cartilage.

Nanomedicines can be taken up by cells via nonspecific and dynamin-dependent (energy-dependent) clathrin and caveolae-mediated endocytosis. While significant effort has focused on targeting pathway-specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin-mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin-independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD-MBA-231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We address unresolved questions of the energetics and mechanism of lipid droplet (LD) biogenesis, and of the role of caveolins in the endoplasmic reticulum (ER) and in mature LDs. LDs are eukaryotic repositories of neutral lipids, which are believed to be synthesised in the ER. We investigate the effects of a curvature-inducing protein, caveolin-1, on the formation and structure of a spontaneously aggregated triolein (TO) lipid lens in a flat lipid bilayer using molecular dynamics (MD) simulations. A truncated form of caveolin-1 (Cav1) localises on the interface between the spontaneously formed TO aggregate and the bulk bilayer, and thins the bilayer at the edge of the aggregate, which may contribute to lowering the energy barrier for pinching off the aggregate from the host bilayer. Simulations of fully mature LDs do not conclusively establish the optimal localisation of Cav1 in LDs, but when Cav1 is in the LD core, the distribution of both neutral lipids in the LD core, and of phospholipids on the engulfing monolayer are altered significantly. Our simulations provide an unprecedented molecular description of the distribution and dynamics of various lipid species in both mature LDs and in the nascent LD inside the bilayer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts' markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.

Type 1 hypokalemic periodic paralysis (HypoPP1) is a poorly understood genetic neuromuscular disease characterized by episodic attacks of paralysis associated with low blood K + The vast majority of HypoPP1 mutations involve the replacement of an arginine by a neutral residue in one of the S4 segments of the α1 subunit of the skeletal muscle voltage-gated Ca 2+ channel, which is thought to generate a pathogenic gating pore current. The V876E HypoPP1 mutation has the peculiarity of being located in the S3 segment of domain III, rather than an S4 segment, raising the question of whether such a mutation induces a gating pore current. Here we successfully transfer cDNAs encoding GFP-tagged human wild-type (WT) and V876E HypoPP1 mutant α1 subunits into mouse muscles by electroporation. The expression profile of these WT and V876E channels shows a regular striated pattern, indicative of their localization in the t-tubule membrane. In addition, L-type Ca 2+ current properties are the same in V876E and WT fibers. However, in the presence of an external solution containing low-Cl - and lacking Na + and K + , V876E fibers display an elevated leak current at negative voltages that is increased by external acidification to a higher extent in V876E fibers, suggesting that the leak current is carried by H + ions. However, in the presence of Tyrode's solution, the rate of change in intracellular pH produced by external acidification was not significantly different in V876E and WT fibers. Simultaneous measurement of intracellular Na + and current in response to Na + readmission in the external solution reveals a rate of Na + influx associated with an inward current, which are both significantly larger in V876E fibers. These data suggest that the V876E mutation generates a gating pore current that carries strong resting Na + inward currents in physiological conditions that are likely responsible for the severe HypoPP1 symptoms associated with this mutation., (© 2017 Fuster et al.)

The Na + ,K + -ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na + ,K + -ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na + ,K + -ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na + ,K + -ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na + ,K + -ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na + ,K + -ATPase mutations and provide information about the interaction of Na + ,K + -ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have established that annexin A2 (ANXA2) is an important factor in the experimental hepatopulmonary syndrome (HPS) serum-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, the detailed mechanism remains unclear. ANXA2 translocated to the caveolin-enriched microdomains (caveolae) in PASMCs upon HPS serum stimulation. The disruption of caveolae by Methyl-β-cyclodextrin (MβCD) alleviated the caveolae recruitment of ANXA2 and the ANXA2-mediated activation of ERK1/2 and NF-κB, so that ANXA2-modulated PASMC proliferation was suppressed. The over-expression of Cav-1 resulted in the relocation of ANXA2 from caveolae and negatively regulated ERK1/2 and NF-κB activation, which inhibited the ANXA2-modulated PASMC proliferative behavior. These data indicate that caveolae function as a signaling platform for ANXA2-induced proliferative behavior and Cav-1 participates upstream of ANXA2 in the activation of ERK1/2 and NF-κB., (Copyright © 2017. Published by Elsevier Inc.)

Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication. IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells., (Copyright © 2017 American Society for Microbiology.)

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P 2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Caveolin-1 (CAV1), the major constituent of caveolae, plays a pivotal role in various cellular biological functions, including cancer and inflammation. The ubiquitin/proteasomal pathway is known to contribute to the regulation of CAV1 expression, but the ubiquitin ligase responsible for CAV1 protein stability remains unidentified. Here we reveal that E3 ubiquitin ligase ZNRF1 modulates CAV1 protein stability to regulate Toll-like receptor (TLR) 4-triggered immune responses. We demonstrate that ZNRF1 physically interacts with CAV1 in response to lipopolysaccharide and mediates ubiquitination and degradation of CAV1. The ZNRF1-CAV1 axis regulates Akt-GSK3β activity upon TLR4 activation, resulting in enhanced production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokine IL-10. Mice with deletion of ZNRF1 in their hematopoietic cells display increased resistance to endotoxic and polymicrobial septic shock due to attenuated inflammation. Our study defines ZNRF1 as a regulator of TLR4-induced inflammatory responses and reveals another mechanism for the regulation of TLR4 signalling through CAV1.

We previously demonstrated that CB 1 receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB 1 receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB 1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB 1 receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB 1 receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB 1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB 1 receptor localization and function., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Liver injury leads to a vasculopathy in which post-translational modifications of endothelial nitric oxide synthase (eNOS) lead to impaired nitric oxide synthesis. We hypothesized that caveolin 1 (CAV1), a well-known eNOS interactor, regulates eNOS activity in sinusoidal endothelial cells (SECs) via its interaction with G-protein-coupled receptor kinase-2 (GRK2) that also post-translationally modifies eNOS. Liver injury with portal hypertension was established using bile duct ligation in rats. CAV1 function was modified using a CAV1 scaffolding domain construct and cDNAs encoding wild-type CAV1, and CAV1 phosphorylation was increased in injured SECs, resulting in increased GRK2-CAV1 interaction and decreased eNOS activity. In injured SECs, endothelin-1 blocked CAV1 phosphorylation induced by CAV1 scaffolding domain, indicating that CAV1 interaction with GRK2 is inversely regulated by endothelin-1 and CAV1 scaffolding domain after liver injury. In addition, after transduction with DNA encoding wild-type CAV1 into SECs isolated from Cav1-deficient mice, GRK2 association with CAV1 was evident, whereas transduction with a dominant negative CAV1 mutated at tyrosine 14 reduced the interaction. Finally, isoproterenol-induced GRK2 phosphorylation enhanced CAV1-GRK2 interaction and reduced eNOS activity. Our data suggest a novel mechanism and model in which CAV1 phosphorylation facilitates CAV1 scaffolding and GRK2-CAV1 interaction, thus clustering eNOS within a complex that inhibits eNOS activity. This process takes place in injured, but not in normal, SECs., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca 2+ -ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca 2+ overload. The decrease of eNOS has been due not only to its Ca 2+ -mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain. These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Aberrant oxidative metabolism is one of the hallmarks of cancer. Reactive species overproduction could promote carcinogenesis via inducing genetic mutations and activating oncogenic pathways, and thus, antioxidant therapy was considered as an important strategy for cancer prevention and treatment. Caveolin-1 (Cav-1), a constituent protein of caveolae, has been shown to mediate tumorigenesis and progression through oxidative stress modulation recently. Reactive species could modulate the expression, degradation, posttranslational modifications, and membrane trafficking of Cav-1, while Cav-1-targeted treatments could scavenge the reactive species. More importantly, emerging evidences have indicated that multiple antioxidants could exert antitumor activities in cancer cells and protective activities in normal cells by modulating the Cav-1 pathway. Altogether, these findings indicate that Cav-1 may be a promising oxidative stress-related target for cancer antioxidant prevention. Elucidating the underlying interaction mechanisms between oxidative stress and Cav-1 is helpful for enhancing the preventive effects of antioxidants on cancer, for improving clinical outcomes of antioxidant-related therapeutics in cancer patients, and for developing Cav-1 targeted drugs. Herein, we summarize the available evidence of the roles of Cav-1 and oxidative stress in tumorigenesis and development and shed novel light on designing strategies for cancer prevention or treatment by utilizing the interaction mode between Cav-1 and oxidative stress.

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture., Competing Interests: The authors declare no conflict of interest.

Caveolin-1 (CAV1) is an oncogenic membrane protein associated with endocytosis, extracellular matrix organisation, cholesterol distribution, cell migration and signaling. Recent studies reveal that CAV1 is involved in metabolic alterations - a critical strategy adopted by cancer cells to their survival advantage. Consequently, research findings suggest that CAV1, which is altered in several cancer types, influences tumour development or progression by controlling metabolism. Understanding the molecular interplay between CAV1 and metabolism could help uncover druggable metabolic targets or pathways of clinical relevance in cancer therapy. Here we review from a cancer perspective, the findings that CAV1 modulates cell metabolism with a focus on glycolysis, mitochondrial bioenergetics, glutaminolysis, fatty acid metabolism, and autophagy.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia., Competing Interests: The authors declare no conflict of interest.

Caveolin-1 is the principle membrane protein of caveolae and plays an important role in various cellular processes. The protein contains two helices (H1 and H2) connected by a three-residue break. Although caveolin-1 is assumed to adopt a U-shaped conformation in the transmembrane domain, with both the N-terminus and C-terminus exposed to the cytoplasm, the structure and dynamics of caveolin-1 in membranes are still unclear. Here, we performed six molecular dynamics simulations to characterize the structure and dynamics of caveolin-1 (residues D82-S136; Cav182-136) in a caveolae-mimicking asymmetric lipid bilayer. The simulations reveal that the structure of the caveolin scaffolding domain of caveolin-1 is dynamic, as it could be either fully helical or partly unstructured. Cav182-136 inserts into the inner leaflet of the asymmetric lipid bilayer with a stable U-shaped conformation and orients almost vertical to the bilayer surface. The simulations also provide new insights into the effects of caveolin-1 on the morphology of caveolae and the possible interacting site of cholesterol on caveolin-1.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1., Competing Interests: The authors declare no conflicts of interest.

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

Caveolin-1 is a membrane protein that possesses an unusual topology where both N- and C-termini are cytoplasmic as a result of a membrane-embedded turn. In particular, proline 110 has been postulated to be the linchpin of this unusual motif. Using a caveolin-1 construct (residues 62-178) reconstituted into dodecylphosphocholine micelles with and without a cholesterol mimic, the changes that occurred upon P110A mutation were probed. Using far UV circular dichroism spectroscopy it was shown that cholesterol attenuated the helicity of caveolin-1, and that mutation of P110 to alanine caused a significant increase in the α-helicity of the protein. Near UV circular dichroism spectroscopy showed significant changes in structure and/or environment upon mutation that again were modulated by the presence of cholesterol. Stern-Volmer quenching and λ(max) analysis of tryptophan residues showed that the proline mutation caused W85 to become more exposed, W98 and W115 to become less exposed, and W128 showed no change. This finding provided evidence that regions proximal and far away from the proline are buried differentially upon its mutation and therefore this residue is strongly tied to maintaining the hydrophobic coverage along the caveolin-1 sequence. In the presence of cholesterol, the accessibilities of the two tryptophan residues that proceeded position 110 were altered much more significantly upon P110A mutation than the two tryptophans aft P110. Overall, this work provides strong evidence that proline 110 is critical for maintaining both the structure and hydrophobic coverage of caveolin-1 and that cholesterol also plays a significant role in modulating these parameters., (Copyright © 2016 Elsevier B.V. All rights reserved)