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Hepatitis C virus (HCV) non-structural 5A (NS5A) protein is associated with a wide variety of host signaling pathways by binding to C-terminal polyproline (PxxP) motifs of various proteins. In this study, we used yeast two-hybrid analysis and a GST pull-down assay to screen a novel NS5A interacting protein and elucidate the binding site and cellular signaling by focusing on recombinant human epidermal growth factor (rhEGF)-mediated ERK1/2 activation. Screening a liver cDNA library revealed that h-prune, a member of the DHH (Asp-His-His) protein superfamily, directly interacted with HCV NS5A C-terminus. In particular, a mutation of five proline amino acids to alanine in this region revealed that these two proteins produced strong interaction through this domain. It is known that h-prune possesses a highly conserved DHH motif, which has exopolyphosphatase activity that accelerates hydrolysis of inorganic polyphosphate. A time-chasing analysis after rhEGF treatment demonstrated that h-prune overexpression almost restored NS5A-mediated attenuation of ERK1/2 phosphorylation, but h-prune itself did not alter this signaling. Although the detailed mechanisms need to be clarified, this study demonstrates that h-prune interacts directly with the PxxP motif of the HCV NS5A C-terminus and that this binding alters the rhEGF-mediated ERK1/2 signaling cascade in liver cells.

Genetic heterogeneity in the nef genes from human immunodeficiency virus type 1 (HIV-1)-infected rapid progressors (RPs) and long-term nonprogressors (LTNPs) was analyzed to identify various amino acid substitutions responsible for the discernible difference in disease progression. It was found that the majority of the strains characterized belonged to subtype C, followed by several BC recombinants and subtype A1. Complete nef subtype C sequences from 33 RPs and seven LTNPs were compared, and it was observed that, in the majority of the sequences from both groups, highly conserved functional motifs showed subtle changes. However, drastic changes were observed in two isolates from LTNPs where the arginine cluster was deleted, while in one of them, additionally, acidic residues were replaced by basic residues (EEEEE→RK(R)KKE). The deletion of the arginine cluster and the mutation of acidic residues to basic residues are predicted to delay disease development by abolishing CD4 downmodulation and causing diminution of major histocompatibility complex class I (MHC-I) downregulation, respectively. Nonetheless, this is an exclusive finding in these LTNPs, which necessitates their analysis at the functional level. The synonymous-to-nonsynonymous substitution ratio was greater than one in both of the groups, suggesting amino acid sequence conservation and functional robustness. Interpatient nucleotide distance within the group and between the two groups showed very little variation, confirming genetic relatedness among isolates.

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl1. p73, a member of the p53 family of tumour-suppressor proteins2,3, can also induce apoptosis3. Here we show that the apoptotic activity of p73α requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, and this binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl tophosphorylate p73 is markedly increased by γ-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.

Activation of the p53 tumor suppressor protein can lead to either cell cycle arrest or apoptosis. Several functional domains necessary for mediating cell cycle arrest and apoptosis in p53 have been mapped, e.g., the proline-rich domain. The proline-rich domain is located within residues 60-90, which comprise five PXXP motifs (where P represents proline and X any amino acid). To further delineate the function of the proline-rich domain and its potential role in transactivation, we generated several groups of cell lines that inducibly express various p53 mutants using a tetracycline-regulated expression system. We found that p53(delta62-91), which lacks all five PXXP motifs in human p53, is capable of inducing cell cycle arrest but not apoptosis, while p53(gln22-ser23/delta62-91), which contains a double point mutation in the activation domain as well as deletion of the proline-rich domain, completely loses its activity. However, p53(delta74-91), which contains only one PXXP motif at its N-terminus, is not only capable of inducing cell cycle arrest but also retains a partial apoptotic activity. Furthermore, we found that deletion of the proline-rich region has no or very mild effects on activation of several transiently transfected p53 target gene promoters, i.e., the p21, MDM2, BAX, and GADD45 promoters. However, such deletion differentially affects p53 induction of endogenous target genes, i.e., induction of p21, MDM2, BTG2, p85, PIG3, PIG6 and PIG11 was reduced or abrogated but induction of BAX, KILLER/DR5, PIG2, PIG7 and PIG8 was not substantially affected. Interestingly, induction of GADD45 was enhanced. These results suggest that the proline-rich region may play a role in chromatin remodeling, which counteracts chromatin-mediated repression for some of the endogenous p53 target genes.

The Src-homology-3 (SH3) domains of the Caenorhabditis elegans protein SEM-5 and its human and Drosophila homologues, Grb2 and Drk (refs 1-4), bind proline-rich sequences found in the nucleotide-exchange factor Sos as part of their proposed function linking receptor tyrosine kinase activation to Ras activation. Here we report the crystal structure at 2.0 A resolution of the carboxy-terminal SH3 domain from SEM-5 complexed to the mSos-derived amino-acid sequence PPPVPPRRR. The peptide is found to bind in an orientation ('minus') that is precisely opposite to that observed previously ('plus' orientation) in other SH3-peptide complexes. This novel ability of peptide-recognition proteins to recognize peptides in two distinct modes may play an important role in the signalling specificity of pathways involving SH3 domains. Comparison of this structure with other SH3 complexes reveals how a conserved binding face can be used to recognize peptides in different orientations, and why the Sos peptide binds in this particular orientation.

BackgroundProtein interactions are essential for most cellular functions. Interactions mediated by domains that appear in a large number of proteins are of particular interest since they are expected to have an impact on diversities of cellular processes such as signal transduction and immune response. Many well represented domains recognize and bind to primary sequences less than 10 amino acids in length called Short Linear Motifs (SLiMs).ResultsIn this study, we systematically studied the evolutionary conservation of SLiMs recognized by SH2, SH3 and Ser/Thr Kinase domains in both ordered and disordered protein regions. Disordered protein regions are protein sequences that lack a fixed three-dimensional structure under putatively native conditions. We find that, in all these domains examined, SLiMs are more conserved in disordered regions. This trend is more evident in those protein functional groups that are frequently reported to interact with specific domains.ConclusionThe correlation between SLiM conservation with disorder prediction demonstrates that functional SLiMs recognized by each domain occur more often in disordered as compared to structured regions of proteins.