ADCK3, an ancestral kinase, is mutated in a form of recessive ataxia associated with coenzyme Q(10) deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphomositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.

A novel locus for split-hand/foot malformation associated with tibial hemimelia (SHFLD syndrome) maps to chromosome region 17p13.1-17p13.3

Split-hand/foot malformation (SHFM) associated with aplasia of long bones, SHFLD syndrome or Tibial hemimelia-ectrodactyly syndrome is a rare condition with autosomal dominant inheritance, reduced penetrance and an incidence estimated to be about 1 in 1,000,000 liveborns. To date, three chromosomal regions have been reported as strong candidates for harboring SHFLD syndrome genes: 1q42.2-q43, 6q14.1 and 2q14.2. We characterized the phenotype of nine affected individuals from a large family with the aim of mapping the causative gene. Among the nine affected patients, four had only SHFM of the hands and no tibial defects, three had both defects and two had only unilateral tibial hemimelia. In keeping with previous publications of this and other families, there was clear evidence of both variable expression and incomplete penetrance, the latter bearing hallmarks of anticipation. Segregation analysis and multipoint Lod scores calculations (maximum Lod score of 5.03 using the LINKMAP software) using all potentially informative family members, both affected and unaffected, identified the chromosomal region 17p13.1-17p13.3 as the best and only candidate for harboring a novel mutated gene responsible for the syndrome in this family. The candidate gene CRK located within this region was sequenced but no pathogenic mutation was detected.

Defective transcription/repair factor IN recruitment to specific UV lesions in trichothiodystrophy syndrome

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFHH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 64PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some mutations affect the recruitment of TFHH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFHH complexes carrying an NH2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFUH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFHH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

p53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that It sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct Importance of DNA repair is hard to access. Here, it is shown that the Induction of photoproducts by UV light (UV-C) significantly Induces apoptosis In a p53-mutated glioma background. This Is caused by a reduced level of photoproduct repair, resulting In the persistence of DNA lesions in p53-mutated glioma cells. UV-C-Induced apoptosis in p53 mutant glioma cells Is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results Indicate that UV-C-induced apoptosis of p53 mutant glioma cells Is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data Indicate that unrepaired DNA lesions Induce apoptosis In p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that Induce the formation of DNA lesions whose global genomic repair is dependent on p53. (Mol Cancer Res 2009;7(2):237-46)

SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Screening of ARHSP-TCC Patients Expands the Spectrum of SPG11 Mutations and Includes a Large Scale Gene Deletion

Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP-TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP-TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice-site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing. (C) 2008 Wiley-Liss, Inc.

pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce`s disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.