Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library.

Many naturally occurring point mutations in the p53 gene lead to a proportion of the encoded protein molecules adopting a distinct, "mutant" conformation characterized by exposure of a normally cryptic epitope recognized by the monoclonal antibody PAb240. Here the PAb240 epitope is defined using a filamentous phage epitope library. The hexapeptides displayed by the PAb240-binding phage isolated from the library were all highly related and allowed both direct localization of the epitope and prediction of a specific interaction between PAb240 and Xenopus TFIIIA. This study demonstrates for the first time the power of phage epitope libraries in the precise definition of previously unmapped epitopes. Identification of the PAb240 epitope precisely defines a region of the p53 molecule structurally altered by the mutation-induced conformational shift.     

Chromosomal mapping and genomic organization of an evolutionarily conserved zinc finger gene ZNF277

A novel C2H2 zinc finger gene, ZNF277, has been localized to human chromosome 7q31.1. The gene is encoded by 12 exons in a genomic fragment of >100 kb between the microsatellite markers D7S523 and D7S471, deleted in a number of malignancies. The predicted open reading frame (ORF) of 438 amino acids shows an overall homology of 50% to the putative ORF F46B6.7 of Caenorhabditis elegans. The presence of a 30-amino-acid coiled-coil domain in both the C. elegans ORF F46B6.7 and ZNF277 is suggestive of functional similarities. ESTs for the murine orthologue ZFP277 are found in early embryonic stem cells, 16-cell stage embryo, and blastocysts. The evolutionary conservation and the expression profile suggest ZNF277 to be a critical regulator of development and differentiation.     

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis

The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ～40-residue GPS motif represents an integral part of a much larger ～320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.     

Identification of a highly conserved domain on phytochrome from angiosperms to algae

A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages.     

Localization of a conserved epitope and an azurin-like domain in the H.8 protein of pathogenic Neisseria

The pathogenic neisseriae, Neisseria gonorrhoeae and Neisseria meningitidis, possess an outer membrane protein, H.8, which contains a conserved monoclonal antibody (MAb)-binding epitope in all strains tested. We have cloned and sequenced a meningococcal H.8 gene, and determined the characteristics of the predicted protein. The predicted signal peptide has features characteristic of a prokaryotic lipoprotein. The region at the N-terminal end of the mature protein (39 amino acids) is primarily composed of alanine, glutamate and proline residues arranged in imperfect repeats with the consensus sequence AAEAP. The epitope for H.8 MAb-binding was localized to a 20-amino-acid sequence within this region. The remainder of the predicted amino acid sequence shows extensive homology to azurins, which are small blue copper-binding proteins found in a limited number of species of pathogenic bacteria.     

IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.     

Precise epitope mapping of malaria parasite inhibitory antibodies by TROSY NMR cross-saturation

We have applied NMR cross-saturation with TROSY detection to the problem of precisely mapping conformational epitopes on complete protein antigen molecules. We have investigated complexes of the Fab fragments of two antibodies that have parasite inhibitory activity, bound to the important malaria vaccine candidate antigen, Plasmodium falciparum MSP1(19). The results indicate remarkable overlap between these epitopes for inhibitory antibodies, and will provide a basis for theoretical modeling of the antibody-antigen interface.     

Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases

Inorganic polyphosphate (poly-P) consists of just a chain of phosphate groups linked by high energy bonds. It is found in every organism and is implicated in a wide variety of cellular processes (e.g. phosphate storage, blood coagulation, and pathogenicity). Its metabolism has been studied mainly in bacteria while remaining largely uncharacterized in eukaryotes. It has recently been suggested that poly-P metabolism is connected to that of highly phosphorylated inositol species (inositol pyrophosphates). Inositol pyrophosphates are molecules in which phosphate groups outnumber carbon atoms. Like poly-P they contain high energy bonds and play important roles in cell signaling. Here, we show that budding yeast mutants unable to produce inositol pyrophosphates have undetectable levels of poly-P. Our results suggest a prominent metabolic parallel between these two highly phosphorylated molecules. More importantly, we demonstrate that DDP1, encoding diadenosine and diphosphoinositol phosphohydrolase, possesses a robust poly-P endopolyphosphohydrolase activity. In addition, we prove that this is an evolutionarily conserved feature because mammalian Nudix hydrolase family members, the three Ddp1 homologues in human cells (DIPP1, DIPP2, and DIPP3), are also capable of degrading poly-P.     

Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed.     

RAP55: insights into an evolutionarily conserved protein family

The RAP55 protein family is evolutionarily conserved in eukaryotes. Two highly conserved paralogues, RAP55A and RAP55B, exist in vertebrates; their functional properties and expression patterns remain to be compared. RAP55 proteins share multiple domains: the LSm14 domain, a serine/threonine rich region, an FDF (phenylalanine-aspartate-phenylalanine) motif, an FFD-TFG box and RGG (arginine-glycine-glycine) repeats. Together these domains are responsible for RAP55 proteins participating in translational repression, incorporation into mRNP particles, protein-protein interactions, P-body formation and stress granule localisation. All RAP55A proteins localise to P-body-like complexes either in the germline or in somatic cells. Xenopus laevis RAP55B has been shown to be part of translationally repressed mRNP complexes in early oocytes. Together these findings suggest that this protein family has evolved a common and fundamental role in the control of mRNA translation. Furthermore human RAP55A is an autoantigen detected in the serum of patients with primary biliary cirrhosis (PBC). The link between RAP55A, P-bodies and PBC remains to be elucidated.     

Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.