Mary-Jane Gething

HSP70 Binding Sites in the Tumor Suppressor Protein p53

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly β-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

BiP-binding Sequences in HIV gp160 IMPLICATIONS FOR THE BINDING SPECIFICITY OF BiP

BiP, a resident endoplasmic reticulum member of the HSP70 family of molecular chaperones, associates transiently with a wide variety of newly synthesized exocytotic proteins. In addition to immunoglobulin heavy and light chains, the first natural substrates identified for BiP, a number of viral polypeptides including the human immunodeficiency virus type 1 envelope glycoprotein gp160 interact with BiP during their passage through the endoplasmic reticulum. We have used a computer algorithm developed to predict BiP-binding sites within protein primary sequences to identify sites within gp160 that might mediate its association with BiP. Analysis of the ability of 22 synthetic heptapeptides corresponding to predicted binding sites to stimulate the ATPase activity of BiP or to compete with an unfolded polypeptide for binding to BiP indicated that about half of them are indeed recognized by the chaperone. All of the confirmed binding sites are localized within conserved regions of gp160, suggesting a conserved role for BiP in the folding of gp160. Information on the characteristics of confirmed BiP-binding peptides gained in this and previous studies has been utilized to improve the predictive power of the BiP Score algorithm and to investigate the differences in peptide binding specificities of HSP70 family members.

Protein folding: The difference with prokaryotes

In prokaryotes, the polypeptide chains in which proteins are synthesized only tend to fold into their final, operational form when the chain is complete. In eukaryotes, by contrast, individual domains of a single protein can fold sequentially and independently. This striking finding can help to resolve the puzzle of why prokaryotes tend to rely much more heavily on chaperonin- assisted protein folding than do eukaryotes, but the molecular mechanisms underlying the different treatment of nascent polypeptide chains remain to be defined.

Molecular chaperones: Clasping the prize

The three-dimensional structure of the substrate-binding domain of DnaK, a bacterial Hsp70, shows how such molecular chaperones can be so promiscuous in recognizing different proteins, yet so accurate in discriminating between unfolded and folded forms of their polypeptide substrates.

Presenilin mutants subvert chaperone function.

Mutant presenilin proteins, known to promote the development of Alzheimer’s disease through increased generation of Aβ42 peptides, appear to compound this insult by downregulating the signalling pathway that adjusts levels of molecular chaperones in the endoplasmic reticulum in response to stress.

Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites.

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to beta-strands and alpha-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

ANALYSIS OF REQUIREMENTS FOR CELL MEDIATED LYSIS INTERACTION BETWEEN VIRAL GLYCOPROTEINS AND CELL PLASMA MEMBRANES

Publisher Summary This chapter presents the analysis of requirements for cell-mediated lysis interaction between viral glycoproteins and cell plasma membranes. The specificity of cytotoxic T killer cells generated in mice after infection with certain viruses is directed to antigenic determinants on cell surfaces induced by the viral and by the host genome. The target cells to be lysed have to carry antigens of the sensitizing virus and antigenic products of the K or D region of the major histocompatibility complex compatible with the specificity repertoire of the T-cells. The protease treatment abolishes the activity of both glycoproteins. Trypsin treatment inactivates fusion and hemolysis activity. V8 protease treatment inactivates HN, eliminating haemagglutinin and neuraminidase activities, but it has no effect on F as far as analysis on polyacrylamide gels, immunological analysis, and amino acid composition is concerned. Because virus–cell and cell–cell fusion has to be preceded by virus-cell adsorption, V8 protease treatment eliminates the functional activities of F as well. All three proteases block the activity of Sendai virus to form the target antigen, indicating that either haemagglutinin-neuraminidase or F is active in the process of target antigen formation.