Mitochondria not only generate cellular energy, but also act as the point for cellular decisions leading to apoptosis. The voltage-dependent anion channel (VDAC), as a major mitochondrial outer-membrane transporter, has an important role in energy production by controlling metabolite traffic and is also recognized as a key protein in mitochondria-mediated apoptosis. In this study, the role of VDAC1 in regulating cell survival and death was investigated by silencing endogenous human (h)VDAC1 expression by using a short hairpin RNA (shRNA)-expressing vector. The shRNA effectively down-regulated the expression in human T-REx-293 cells of hVDAC1 but not murine (m)VDAC1. Cells in which hVDAC1 expression was decreased by ≈90% proliferated extremely slowly. Normal growth was, however, restored upon expression of mVDAC1 in a tetracycline-regulated manner. Although low tetracycline concentrations promoted cell growth, high concentrations induced mVDAC1 overexpression, leading to cell death. Cells with low levels of VDAC1 showed 4-fold-lower ATP-synthesis capacity and contained low ATP and ADP levels, with a strong correlation between ATP levels and cell growth, suggesting limited metabolite exchange between mitochondria and cytosol. The possibility of suppressing endogenous hVDAC1 expression and introducing native and mutated mVDAC1 is used to further explore the involvement of VDAC1 in apoptosis. Cells suppressed for hVDAC1 but expressing either native mVDAC1 or an E72Q mutant underwent apoptosis induced by various stimuli that can be inhibited by ruthenium red in the native cells but not in the mutated cells, suggesting that VDAC1 regulates apoptosis independent of the apoptosis-inducing pathway.

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC.[␣-32 P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [␣-32 P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of ϳ17-and ϳ14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotidebinding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.It has recently been recognized that there exists a metabolic coupling between the cytosol and the mitochondria, with the outer mitochondrial membrane (OMM), 2 the boundary between these compartments, fulfilling an important function in this coupling (1-4). In the cross-talk between mitochondria and cytosol, mitochondrial ATP and Ca 2ϩ play major roles. As the primary transporter of nucleotides, Ca 2ϩ , and other ions and metabolites across the OMM (reviewed in Refs. 5 and 6), the voltage-dependent anion channel (VDAC) mediates a substantial portion of the OMM molecular traffic. It has been demonstrated that VDAC is permeable to Ca 2ϩ and possesses Ca 2ϩ -binding sites that control its activity (reviewed in Ref. 5). VDAC permeability is also regulated by associated protein(s) (reviewed in Refs. 5-7), and by different ligands that bind to VDAC, such as glutamate, ruthenium red, La 3ϩ , and NADH (reviewed in Refs. 5 and 6). Thus, by providing the pathway for anions, cations, ATP, and other metabolites into and out of the mitochondria, as well as possessing ligand-binding sites, VDAC plays an important role in the regulation of mitochondrial functions. Indeed, VDAC has been suggested to control OMM permeability, coupled respiration, and cell survival (3,4,8).It has been proposed that activati...

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.