Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

The functional significance of absence: the chromosomal segment harboring Tp53 is absent from the T55 rat radiation hybrid mapping panel

The T55 rat radiation hybrid (RH) mapping panel has been reported to retain the entire rat genome at retention frequencies between 22% and 37%. However, we found that a small segment of rat chromosome 10 harboring at least four different genes, including Tp53, was completely absent from the panel (retention frequency = 0%). Two other markers located in the vicinity exhibited much reduced retention (2-6%). RH clones are generated by transferring highly fragmented DNA into a recipient cell. There might be a strong selection against the transfer and retention of chromosome segments harboring an intact Tp53, as the action of this gene might prevent proliferation and establishment of the RH clone. Our finding further suggests that unexpected low retention or absence of chromosome segments in an RH panel may represent indications that the segments harbor genes with important functions in cell proliferation control.     

Migraine with Aura Susceptibility Locus on Chromosome 19p13 Is Distinct from the Familial Hemiplegic Migraine Locus

Migraine is a common neurological disease with a major genetic component. Recently, it has been proposed that a single locus on chromosome 19p13 contributes to the genetic susceptibility of both rare familial hemiplegic migraine (FHM) and more common types of migraine, migraine with aura and migraine without aura. We analyzed 16 families for co-segregation of migraine with aura and chromosome 19p13 markers. Using multipoint model-free linkage analysis, we obtained a lod score of 4.28 near D19S592. Using an affecteds-only model of linkage, we observed a lod score of 4.79 near D19S592. We were able to provide statistical evidence that this locus on chromosome 19p13 is most likely not the gene CACNA1A, mutations in which cause FHM. These data indicate that chromosome 19p13 contains a locus which contributes to the genetic susceptibility of migraine with aura that is distinct from the FHM locus.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

p53 activation inhibits ochratoxin A-induced apoptosis in monkey and human kidney epithelial cells via suppression of JNK activation

Ochratoxin A (OTA), one of the major food-borne mycotoxins, induces apoptosis in various types of cells. Induction of apoptosis is suggested to be one of the major cellular mechanisms behind OTA-induced diverse toxic effects. However, the molecular mechanisms involved, especially the role of p53 in OTA-induced apoptosis have not been clearly elucidated. In the present study, we find that p53 activation exerts pro-survival function to inhibit apoptosis induction in MARC-145, Vero monkey kidney cells and HEK293 human kidney cells in response to ochratoxin A treatment. We further demonstrate that the pro-survival activity of p53 is attributed to its ability to suppress JNK activation that mediates apoptotic signaling through down-regulation of Bcl-xL. To our knowledge, this is first report of pro-survival role of p53 in OTA-induced apoptosis in kidney epithelial cells. Our findings provide a novel insight into the mechanisms of OTA-induced apoptosis in kidney epithelial cells.     

The role of palmitoylation in directing dopamine D1 receptor internalization through selective endocytic routes

We previously determined that D1 receptors can endocytose through caveolae, a subset of lipid rafts, in addition to internalization via a clathrin-dependent pathway. In this report, we investigated the potential role that palmitoylation might have on directing D1 receptor internalization through either a clathrin or caveolar-dependent route. Through whole cell binding analysis and sucrose gradient fractionation studies, we demonstrated that although palmitoylation of the D1 receptor was not required for agonist-independent localization to caveolae, agonist induced internalization kinetics of a de-palmitoylated D1 receptor were accelerated ∼8-fold in comparison to wild-type D1 receptor and were very similar to that observed for clathrin-dependent D1 receptor internalization. Additionally, inhibition of the clathrin mediated pathway led to significant attenuation in the extent of agonist induced internalization of the de-palmitoylated D1 receptor, suggesting the de-palmitoylated D1 receptor was directed to a clathrin-dependent internalization pathway. Taken together, these data suggest that palmitoylation may be involved in directing agonist-dependent D1 receptor internalization through selective endocytic routes.     

Identification of New Regulatory Sequences Far Upstream of the Mouse Monocyte Chemoattractant Protein-1 Gene

We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.     

RGSZ1 and Ret RGS: Two of Several Splice Variants from the Gene RGS20

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.     

Single-Nucleotide Polymorphism Alleles in the Insulin Receptor Gene Are Associated with Typical Migraine

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.     

Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand–receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.