Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

A homologue of the fungal tetraspanin Pls1 is required for Epichloë festucae expressorium formation and establishment of a mutualistic interaction with Lolium perenne

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium-like structure known as an expressorium. In plant-pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild-type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium-like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring-like structure at the point of cuticle exit as found in the wild-type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

Down-regulation of macrophage CD9 expression by interferon-gamma.

CD9, a member of the tetraspanin family is a cell surface marker expressed on myeloid and nonmyeloid as well as on neoplastic cells. The present study has focused on the role of inflammation and macrophage activation in the regulation of CD9 expression. We report that the expression of CD9 on primary cultures of murine peritoneal macrophages was down regulated by Interferon-gamma, IFN-gamma. This down regulation was concentration-dependent and maximal by 48 h. The changes in surface expression were consistent with similar reductions in CD9 protein and message levels by Western and Northern blot analyses. The mechanism by which IFN-gamma decreases CD9 expression appears to be through the Stat1 signaling pathway as Stat1 knockout mice did not demonstrate any reduction in CD9 expression by IFN-gamma treatment. These results represent the first evidence for the down regulation of CD9 expression with macrophage activation.     

The L6 membrane proteins--a new four-transmembrane superfamily

L6, IL-TMP, and TM4SF5 are cell surface proteins predicted to have four transmembrane domains. Previous sequence analysis led to their assignment as members of the tetraspanin superfamily. In this paper, we identify a new sequence (L6D) that is strikingly similar to L6, IL-TMP, and TM4SF5. Analyses of these four sequences indicate that they are not significantly related to genuine tetraspanins, but instead constitute their own L6 superfamily.     

Recruitment of tetraspanin TSP-15 to epidermal wounds promotes plasma membrane repair in C. elegans

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Glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility

Glycosphingolipids (GSLs) GM3 (NeuAcα3Galβ4Glcβ1Cer) and GM2 (GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer) inhibit (i) cell growth through inhibition of tyrosine kinase associated with growth factor receptor (GFR), (ii) cell adhesion/motility through inhibition of integrin-dependent signaling via Src kinases, or (iii) both cell growth and motility by blocking “cross-talk” between integrins and GFRs. These inhibitory effects are enhanced when GM3 or GM2 are in complex with specific tetraspanins (TSPs) (CD9, CD81, CD82). Processes (i)-(iii) occur through specific organization of GSLs with key molecules (TSPs, caveolins, GFRs, integrins) in the glycosynaptic microdomain. Some of these processes are shared with epithelial-mesenchymal transition induced by TGFβ or under hypoxia, particularly that associated with cancer progression.