Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface
ABSTRACT Palmitoylation of tetraspanins affects protein–protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.
SourceAvailable from: Antonios D. Konitsiotis[Show abstract] [Hide abstract]
ABSTRACT: Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localisation. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmatic reticulum (ER) that palmitoylates Hedgehog proteins, is a member of a small subfamily of MBOAT proteins that acylate secreted proteins, and is an important drug target in cancer. However little is known about HHATs structure and mode of function. We show that HHAT is comprised of 10 transmembrane domains and 2 reentrant loops with the critical His and Asp residues on opposite sides of the ER membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines, which maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesised catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insight into how the protein may function in vivo. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 12/2014; DOI:10.1074/jbc.M114.614578 · 4.60 Impact Factor
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ABSTRACT: Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.Development 02/2015; DOI:10.1242/dev.118844 · 6.27 Impact Factor
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ABSTRACT: Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.PLoS ONE 10/2014; 9(10):e111333. DOI:10.1371/journal.pone.0111333 · 3.53 Impact Factor