Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.

The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum.

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.

The yeast inositol polyphosphate 5-phosphatases inp52p and inp53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations.

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and PtdIns(3, 5)P(2). Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

Presenilin mutants subvert chaperone function.

Mutant presenilin proteins, known to promote the development of Alzheimer's disease through increased generation of Abeta42 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.

Role and regulation of the ER chaperone BiP.

BiP, an HSP70 molecular chaperone located in the lumen of the endoplasmic reticulum (ER), binds newly-synthesized proteins as they are translocated into the ER and maintains them in a state competent for subsequent folding and oligomerization. BiP is also an essential component of the translocation machinery, as well as playing a role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the proteasome. BiP is an abundant protein under all growth conditions, but its synthesis is markedly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER. This attribute provides a marker for disease states that result from misfolding of secretory and transmembrane proteins.

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.

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.

Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP.

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.

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 beta-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.

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.

Normal protein folding machinery.

A highly conserved protein folding machine has been maintained in the cytosol of both prokaryotic and eukaryotic organisms and in eukaryotic mitochondria. Homologous components of this machinery have also been identified in other organelles such as the endoplasmic reticulum in which HSP70 and DnaJ-like homologs reside. The high degree of conservation presumably reflects the proficiency with which these molecules have evolved to mediate the folding of proteins to their native functional states.

BiP binding sequences in antibodies.

During the process of folding and assembly of antibody molecules in the endoplasmic reticulum, immunoglobulin heavy and light chains associate transiently with BiP, a resident endoplasmic reticulum protein that is a member of the Hsp70 family of molecular chaperones. BiP is thought to recognize unfolded or unassembled polypeptides by binding extended sequences of approximately seven amino acids that include bulky hydrophobic residues not normally exposed on the surface of native proteins. We used a computer algorithm developed to predict BiP binding sites within protein primary sequences to identify sites within immunoglobulin chains that might mediate their association with BiP. Very few of the sequential heptapeptides in the heavy or light chain sequences were potential BiP binding sites. Analysis of the ability of synthetic heptapeptides corresponding to 24 potential sites in heavy chains to stimulate the ATPase activity of BiP indicated that at least half of them were authentic BiP binding sequences. These sequences were not confined to a single domain of the heavy chain but were distributed within both the VH and CH domains. Interestingly, when the BiP binding sequences were mapped onto the three-dimensional structure of the Fd antibody fragment, the majority involve residues that participate in contact sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody molecules by binding to hydrophobic surface regions on the isolated immunoglobulin chains that subsequently participate in interchain contacts.

Tissue-type plasminogen activator-induced invasion and metastasis of murine melanomas.

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.

Common and divergent peptide binding specificities of hsp70 molecular chaperones.

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.

The cellular response to unfolded proteins: intercompartmental signaling.

Both prokaryotic and eukaryotic cells respond to the accumulation of unfolded proteins by increasing the transcription of genes encoding molecular chaperones and other stress-responsive proteins. Different sets of genes are activated when particular cellular compartments are burdened with unfolded proteins. Cells thus maintain mechanisms to monitor changes in the concentration of unfolded proteins not only in the cytosol, but also in membrane-bound extracytoplasmic compartments. During the past year, work in yeast has identified a transmembrane receptor that appears to play a pivotal role in the regulation of protein folding. This receptor monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones that are localized in the endoplasmic reticulum. Work using Escherichia coli suggests that prokaryotes also contain an intercompartmental 'unfolded protein' signaling pathway, in this case from the periplasmic space or outer membrane to the cytoplasm.

The pro region of human intestinal lactase-phlorizin hydrolase.

Human small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential cleavage steps: the first in the endoplasmic reticulum to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to mature 160-kDa LPH (denoted LPH beta). The LPH beta molecule is subsequently targetted to the brush-border membrane. Characterization of the N-terminal profragment (denoted LPH alpha) of pro-LPH using an epitope-specific, anti-peptide polyclonal antibody reveals that LPH alpha (i) has an apparent molecular weight of approximately 100,000, (ii) is not associated with LPH beta after cleavage of pro-LPH has occurred, and (iii) is not transported to the cell surface or secreted into the extracellular medium. In biosynthetic labeling experiments, a clear precursor/product relationship could be demonstrated between pro-LPH and the LPH alpha and LPH beta polypeptides. Further, LPH alpha has a significantly shorter half-life than LPH beta. LPH alpha is neither N- nor O-glycosylated, despite the presence of 5 potential N-glycosylation sites. LPH alpha, which is rich in cysteine and hydrophobic amino acid residues, may fold rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. When expressed independently in COS-1 cells, the LPH beta polypeptide forms a misfolded, transport-incompetent molecule. We propose a role for the LPH alpha domain within the pro-LPH molecule as an intramolecular chaperone during folding in the ER.

Interleukin-1 promotes hyperglycemia and insulitis in mice normally resistant to streptozotocin-induced diabetes.

By administering physiological doses of interleukin-1 (IL-1) concurrently with multiple low doses of the beta cell toxin streptozotocin (MSZ), we observed an augmentation of diabetes by IL-1 in four different strains of mice. Augmentation of hyperglycemia by IL-1 was most prominent in the two MSZ-resistant mouse strains Balb/cJ and A/J. Furthermore, concurrent treatment with IL-1 and MSZ rendered these MSZ-resistant mice susceptible to the development of significant insulitis when compared to mice treated with MSZ alone. Development of insulitis was dependent upon the dose of IL-1; it was only observed at an IL-1 dose of 250 ng/kg body weight. Analysis of the leukocytic infiltrate in the islets of mice after treatment with 250 ng/kg IL-1 plus MSZ revealed the presence of L3T4+ and Lyt-2+ T lymphocytes. Administration of MSZ alone or IL-1 alone did not produce diabetes in the MSZ-resistant mice, indicating that neither of these agents was toxic to the beta cells by itself. We conclude that IL-1 synergizes with MSZ to augment diabetes in mice that are normally resistant to the diabetogenic effects of MSZ.

Expression of lactase-phlorizin hydrolase in sheep is regulated at the RNA level.

Lactase-phlorizin hydrolase (LPH) is expressed on the intestinal brush border and is responsible for the hydrolysis of lactose, the chief sugar in mammalian milk. The enzyme activity of LPH peaks soon after birth in most mammals and declines to much lower levels before adolescence. The molecular basis of this pattern of expression has not been clearly established. We have measured relative amounts of LPH mRNA in intestine from sheep with ages across a developmental spectrum, including third trimester fetal lambs, newborn lambs and adult sheep. LPH mRNA levels in the jejunum decline approximately 50-fold between infancy and adulthood, in parallel with the reduction in both lactase specific activity and immunologically reactive lactase protein expression in sheep jejunum. LPH mRNA is present in high concentration in the duodenum of newborn lambs, but steadily declines by day 34 and is dramatically reduced in adults. Because the changes in LPH mRNA, protein, and enzymic activity are generally parallel, we conclude that the developmental regulation of LPH in sheep is probably mediated primarily at the mRNA level.