Bcs1p can rescue a large and productive cytochrome bc1 complex assembly intermediate in the inner membrane of yeast mitochondria

The yeast cytochrome bc₁ complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc₁ complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc₁ assembly and the formation of a functionally inactive bc₁ core structure of about 500-kDa. This immature bc₁ core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc₁ core structure leading to the formation of the functional homodimeric bc₁ complex. Following Bcs1p expression, the mature bc₁ complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc₁ complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc₁ complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc₁ core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc₁ complex and gives new insights into the molecular mechanisms involved in the last steps of bc₁ assembly.     

Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria?

A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented.     

Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.     

Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4A resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall.     

Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.     

HLA association with nasopharyngeal carcinoma in southern Tunisia

In order to study the association of HLA-A, -B and/or DRB1, DQB1 and the nasopharyngeal carcinoma (NPC), 141 patients affected with NPC were typed for the HLA class I by serology method of microlymphocytotoxicity. Among these patients 101 were genotyped for HLA class II system by the PCR-SSP technique. HLA typing results were compared to those of 116 controls. We found that the HLA-A31 and -A33 antigens were significantly more expressed in patients than in the controls (P = 0.016 and 0.010, respectively) and the HLA-A19 antigen, was significantly more frequent in patients when compared to the controls (P = 0.007). The HLA-DRB1*03 and DRB1*13 alleles were significantly more frequent in patients as compared to the controls. The DRB1*01 allele was expressed with a frequency of 20.69% in the controls whereas it was only detected in 3.96% of the NPC patients. Furthermore, the DQB1*05 allele was expressed at a frequency which was significantly less important in affected patient (P = 0.03), whereas, the DQB1*02 allele was more frequent in patients (P = 0.643 × 10⁻⁴). Thus our study revealed a significant increase of HLA-A31, A33, A19, B16, B53 and DRB1*03, DRB1*13 and DQB1*02 alleles in our patients. These markers could play a predisposing role in the development of NPC. In contrast, a decrease of HLA-B14, -B35 and DRB1*01 and DQB1*05 alleles was found suggesting a likely protective effect.     

First molecular diagnosis of Donohue syndrome in Africa: novel unusual insertion/deletion mutation in the <Emphasis Type="Italic">INSR</Emphasis> gene

Donohue syndrome (DS) is a very rare autosomal recessive disease affecting less than one in a million life births. It represents the most severe form of insulin resistance due to mutations involving the insulin receptor (IR) gene “INSR”. DS is characterized by pre- and postnatal growth retardation with failure-to-thrive, lipoatrophy, acanthosis nigricans, hypertrichosis, and dysmorphic features. An exhaustive INSR gene sequencing was performed after PCR amplification of coding exons and introns boundaries. Bioinformatic tools, including ESEfinder, MFOLD and Proter software were also used to predict the impact of INSR mutation on INSR on gene expression as well as on the protein structure and function. The results have shown a novel unusual c.3003_3012delinsGGAAG (p.S1001_D1004delinsRE) insertion/deletion (indel) mutation within the exon 16 in the three patients, which represent the fourth indel mutation within the INSR gene. The mutation modifies the secondary structure of DNA and RNA, as well as the composition of exonic splicing enhancers of exon 16. Moreover, despite the conservation of the secondary structure of the IR, the p.S1001_D1004delinsRE in-frame mutation is accompanied by the loss of four amino acids replaced by two residues of different nature and hydrophobicity level in the juxtamembrane domain of the receptor. The results have confirmed the role of the juxtamembrane domain of IR involved in a crucial interaction of the IR with cellular effectors essentially the IR substrate 1 (IRS-1), the SHC and the Nck proteins that ensure the signal mediated by the insulin transduction pathway in target cells. Our findings have also proven the genotype/phenotype correlation between INSR mutation and DS phenotype severity.     

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locus-specific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.     

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.     

Evaluation of the salivary proteome as a surrogate tissue for systems biology approaches to understanding appetite

Current measurement of appetite depends upon tools that are either subjective (visual analogue scales), or invasive (blood). Saliva is increasingly recognised as a valuable resource for biomarker analysis. Proteomics workflows may provide alternative means for the assessment of appetitive response. The study aimed to assess the potential value of the salivary proteome to detect novel biomarkers of appetite using an iTRAQ-based workflow. Diurnal variation of salivary protein concentrations was assessed. A randomised, controlled, crossover study examined the effects on the salivary proteome of isocaloric doses of various long chain fatty acid (LCFA) oil emulsions compared to no treatment (NT). Fasted males provided saliva samples before and following NT or dosing with LCFA emulsions. The oil component of the DHA emulsion contained predominantly docosahexaenoic acid and the oil component of OA contained predominantly oleic acid. Several proteins were present in significantly (p<0.05) different quantities in saliva samples taken following treatments compared to fasting samples. DHA caused alterations in thioredoxin and serpin B4 relative to OA and NT. A further study evaluated energy intake (EI) in response to LCFA in conjunction with subjective appetite scoring. DHA was associated with significantly lower EI relative to NT and OA (p=0.039). The collective data suggest investigation of salivary proteome may be of value in appetitive response. This article is part of a Special Issue entitled: Proteomics: The clinical link.