Evolutionary conservation of biogenesis of beta-barrel membrane proteins

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.     

Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.     

A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides

Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.     

线粒体膜间隙蛋白质释放与细胞凋亡研究进展

近年来,国内外有关线粒体和肿瘤的关系研究的越来越多,开发与线粒体相关的新型药物并诱导肿瘤细胞凋亡逐渐成为热点.该文主要综述了近年来关于几种线粒体膜间隙蛋白质在细胞凋亡中的作用和由线粒体中释放的可能机制及其在肿瘤药物治疗中的最新进展,指出线粒体膜间隙蛋白质在诱导肿瘤细胞凋亡中具有重要作用.     

Superoxide activates mitochondrial uncoupling proteins.

Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.     

四螺旋桨家族蛋白质序列——结构关系研究

蛋白质的三级结构唯一地由其氨基酸序列决定,这是广为人所接受的。然而,很多具有规则三级结构的蛋白质其氨基酸序列接近随机,这使得人们感到很困惑。本文将以四螺旋桨家族蛋白质为例,通过简化氨基酸残基,根据相似性方法把序列中的隐含对称性显示出来。结果表明氨基酸序列中的隐含对称性与三级结构的四重准对称性一致。     

New variation on the translocation of proteins during early biogenesis of apolipoprotein B

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the endoplasmic reticulum, nascent secretory proteins must be translocated through an aqueous channel in the membrane into the aqueous lumen, so some novel form of processing may be necessary to maintain the solubility of apo B during its translocation. We have discovered that the biogenesis of apo B in cell-free systems does indeed involve a new variation on protein translocation: unlike typical secretory proteins, apo B is synthesized as a series of transmembrane chains with large cytoplasmic domains and progressively longer amino-terminal regions that are protected against added proteases during the translocation process. In contrast to typical transmembrane proteins, these transmembrane chains are not integrated into the bilayer. Moreover, the transmembrane chains with the shortest protected domains are precursors of forms whose protection is progressively extended to cover the length of the protein. This stepwise conversion occurs post-translationally for the most part. We propose a model on the basis of these findings for the biogenesis of apo B.     

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.     

The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.     

Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins

By analysis of a temperature-sensitive yeast mutant, a heat-shock protein in the matrix of mitochondria, mitochondrial hsp70 (Ssc1p), is found to be involved both in translocation of nuclear-encoded precursor proteins across the mitochondrial membranes and in (re)folding of imported proteins in the matrix.     

过表达蛋白合成与分泌相关蛋白提高CHO细胞anti-hLAG3产量

对比研究了过表达蛋白合成与分泌途径中的20种蛋白对瞬时表达细胞（ExpiCHO-S细胞）表达anti-hLAG3产量的影响,考察了转录因子XBP1s对ExpiCHO-S细胞生长、抗体产量和抗体亲和力的影响。结果表明,当所研究蛋白的表达质粒与anti-hLAG3质粒以1：4的质量比共转染时,XBP1s能够使抗体产量提高约30%,GADD34、NFKBIz、CERT、C/EBPα和mTOR的过表达则会明显降低抗体产量,其他14种蛋白对抗体产量的影响较小。随着XBP1s共转染比例的增加,其对anti-hLAG3生产的促进作用增强,当共转染比例提高到60%时,相对于共转染比例为60%的EGFP,可使转染后168 h的抗体产量提高到2.1倍。此外,XBP1s的过表达对活细胞密度和细胞活率以及抗体亲和力的影响均较小。以上结果表明XBP1s可作为细胞工程的一个有效靶标用于提高CHO细胞重组蛋白的生产性能。     

Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins

The mitochondrial import receptors MOM19 and MOM72 form a complex with two other proteins of the mitochondrial outer membrane, MOM38 and MOM22. This receptor complex is involved in recognition, membrane insertion and translocation of precursor proteins with MOM38 constituting (at least part of) the general insertion site GIP.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins

The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.