Membrane Rearrangements And Protein Refolding In Biologi

Child Health And Human Development

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Abstract

The ability of membrane lipids to form continuous and closed bilayers is essential for many functions of biological membranes. However, many biological processes involve local and transient breaking of membrane continuity. In particular, protein-mediated membrane fusion is an important stage of exocytosis, protein trafficking, fertilization and enveloped virus infection. We are interested in the mechanisms by which specialized proteins reshape and remodel the membranes. Our recent work has established the striking similarity of membrane rearrangement pathways in biological fusion driven by fusion proteins as diverse as alphavirus E1 and influenza hemagglutinin. In our search for the ways to control fusion reactions during viral entry, we concentrated on an unexplored stage of viral fusion. Virus-cell membrane contact is crowded with proteins (including fusion proteins and receptors) that cover both of the membranes. To allow tight bilayer contact, hemifusion, and fusion pore opening, membrane proteins have to be displaced from the future fusion site. We found the way to block this stage by crosslinking surface glycoproteins. Moreover, we found that several antiviral lectin components of the innate immunity block influenza virus infection by this novel mechanism. In another project aimed at elucidation of the mechanisms by which cationic peptides deliver macromolecules into cells, we explored the contributions of clathrin-dependent endocytosis and heparan sulfate receptors. 1. Influenza and Sindbis viruses are among the best-studied prototypes of fusion machinery. For both viruses, fusion is triggered by acidification of the virus-containing endosome. While the final lowest-energy forms of Sindbis E1, influenza virus hemagglutinin (HA) and many other fusion proteins share an important motif, two sequences that interact with membranes: the fusion peptide and the transmembrane domain relocate to the same end of the rod-like molecule. However, HA and E1 differ radically in their initial structures and have come to represent two divergent classes of viral fusion proteins. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low-pH triggered E1- mediated cell-cell fusion by reducing the numbers of activated E1 proteins and by blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small, and then expanding, fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. Thus fusion proteins of both classes (I and II) drive the entire fusion pathway, rather than merely catalyze the merger of the contacting monolayers of two membranes. Our finding that dissimilar viral fusion proteins catalyze fusion-through-hemifusion pathway points to universality of this mechanism of biological fusion. The results also support the hypothesis that the final hairpin structure shared by diverse viral fusion proteins and by proteins involved in intracellular fusion is more important for fusion than the initial metastable conformations of these proteins. At the very early stage of biological fusion proteins have to be displaced from the prospective fusion site. We explored the importance of this fusion stage in antiviral activity of retrocyclin-2 (RC2). RC2 is a circular octadecapeptide with three disulphide bonds. It belongs to the family of theta-defensins - antimicrobial peptides expressed by leukocytes and by epithelial cells that protect the host against microorganisms. As other defensins, RC2 has a broad-range antimicrobial activity. We found that RC2 inhibited viral entry by blocking membrane fusion. Using fluorescence recovery after photobleaching assay we found that RC2, a multivalent lectin, immobilizes membrane glycoproteins. Thus, in contrast to fusion inhibitors that specifically target fusion proteins, receptors, or bilayers, RC2 prevented HA-mediated fusion by erecting a network of crosslinked and immobilized surface glycoproteins. The lectin-activity that underlies the fusion-inhibiting activity of RC2 has also been described for other important components of the innate immune system including mannan-binding lectin (MBL) and human beta defensin-3 (HBD3). We found human MBL and HBD3 to have fusion- and membrane protein mobility- inhibiting activities similar to those observed for RC2. Crosslinking of surface glycoproteins by endogenous lectin-like host defense molecules apparently blocks an essential but hitherto unexplored stage of viral entry: displacement of proteins from the prospective fusion site. 2. Recent advances in identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA and small interfering RNA, for molecular therapy. Cationic cell-penterating peptides (CPP) such as TAT peptide derived from the protein transduction domain of HIV TAT protein are widely considered as a promising approach for delivery of proteins and nucleic acids into cells. We examined the mechanisms by which TAT peptide enters living cells. We found that TAT entry into several different primary cells as well as in many stable cell lines to be ATP- and temperature- dependent indicating the involvement of endocytosis. Judging from the effects of specific inhibitors, entry of the unconjugated TAT peptide enters cells involves a clathrin-dependent endocytic pathway. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. To evaluate the role of heparan sulfates in the TAT uptake, we used mutant cells lacking surface heparan sulfate (pgs-A745 and pgs-D677 cell lines) and cell pre-treatments with heparinase III. Uptake inhibition under both conditions indicated the importance of heparan sulfate receptors for the uptake of TAT peptide in wild type CHO cells. However TAT internalization in the absence of heparan sulfate proteoglycans, indicating the existence of heparan sulfate independent mechanisms of entry. Thus, whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. These results along with the recent literature indicate that the uptake of CPPs and their cargo in different cells involves different receptors and different types of endocytosis clathrin-dependent endocytosis, raft/caveolin-dependent endocytosis and macropinocytosis.