Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential.

BACKGROUND INFORMATION: Human OPA1 (optic atrophy type 1) is a dynamin-related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. RESULTS: We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L-OPA1 (long isoforms of OPA1) and three S-OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L-OPA1 to S-OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy-metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins-associated rhomboid-like protein) - the human orthologue of Pcp1/Rbd1 - and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix-oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock-down of YME1L (human yme1-like protein), an IMS-oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of DeltaPsim (inner mitochondrial membrane potential) or OPA1 processing. CONCLUSIONS: Metalloprotease-mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

S1 constrains S4 in the voltage sensor domain of Kv7.1 K+ channels

Voltage-gated K(+) channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (approximately 190 degrees) and outward translation of S4 (approximately 12 A) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1-S4 interaction to an inter-VSD S1-S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating.     

Phospholipid Asymmetry in Acetylcholine Receptor Clusters

We modified the lipids of rat myotubes in tissue culture to determine the transmembrane orientation of aminophospholipids in clusters of acetylcholine receptors (AChR). Trinitrobenzenesulfonic acid and N -hydroxysuccinimidobiotin were used to modify the amino groups of phospholipids. Reaction conditions were selected to prevent penetration of the chemical probes into the cell interior. Fluorescence microscopy was used to confirm that the probes remained impermeant. Analysis of aminophospholipids associated with clusters isolated from chemically modified cells and comparisons to results of chemically modifying isolated acetylcholine receptor clusters indicated that at least 77% of plasma membrane aminophospholipids was located in the interior leaflet of the lipid bilayer. We address the possibility that aminophospholipids on the inner lipid leaflet may contribute to the association between the cytoskeleton and the membrane at AChR clusters.     

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.     

Separate fusion of outer and inner mitochondrial membranes

Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately.     

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A₋₃ = 42%, G₋₃ = 36% and G₊₄ = 47%), only 37.4% of the genes adhere to the purine (R)₋₃/G₊₄ rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R₋₃ and G₊₄, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A₊₁T₊₂G₊₃ initiation codons adhered much more strongly to the R₋₃/G₊₄ rule. Among the intron-less histone genes, only the H3 genes adhere to the R₋₃/G₊₄ rule, while the H1, H2A, H2B and H4 genes usually lack either R₋₃ or G₊₄. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A₊₁→T₊₁ mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.     

Histological examination of low temperatures or TIBA-induced swelling of pepper ovaries

The ovaries of pepper (Capsicum annum L.) plants grown under low night temperatures (12 °C) were larger than those of plants grown under high night temperatures (18 °C). TIBA, applied to young flower buds grown under the higher temperature regime markedly stimulated ovary swelling, similarly to the effect of low night temperatures, whereas, NAA had a much smaller effect. STS or AOA did not reverse or attenuate the effect of either low temperatures or of TIBA. Histological examination of various tissues across the ovaries revealed marked increases in transverse and longitudinal diameters of cell of the swelled ovaries, concomitantly with a smaller increase in the number of cells in the receptacle and placenta. It is suggested that the swelling induced by low night temperatures or TIBA results mostly from the enlargement of all the ovary cells, and only to a smaller extent, by an increase in cell number in some of the ovary tissues. Since TIBA, an auxin transport inhibitor, causes auxin accumulation in the treated organ – in this case the developing ovary – and the auxin NAA stimulated ovary growth, one may conclude that auxins are involved in the observed ovary enlargement, whereas ethylene has no role in this phenomenon.     

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.