The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

Interactions between M protein and other structural proteins of severe,acute respiratory syndrome-associated coronavirus

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP

Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

Purification and structural analysis of a murine chemotactic cytokine (CP-10) with sequence homology to S100 proteins.

In delayed-type hypersensitivity reactions, cytokine-mediated cell migration leads to localized accumulation of neutrophils and mononuclear cells over 4-48 h. In contrast to transient (2-6 h) responses elicited by other chemotactic factors, earlier studies indicated that a chemotactic activity previously described in our laboratory elicited skin test responses over 24 h, identical to those induced by injection of antigen into a sensitized test subject. We have isolated this factor, a 10.3-kDa chemotactic protein designated CP-10, from supernatants of activated murine spleen cells. Purification to homogeneity was achieved using affinity chromatography on iminodiacetic acid-immobilized copper and cation-exchange, mixed mode (cation exchange/metal affinity), reversed-phase, and size-exclusion high performance liquid chromatography. CP-10 had maximal chemotactic activity for neutrophils at 10(-13) M. The 76-amino acid sequence, obtained by automated N-terminal microsequence analysis of native CP-10, and fragments derived from trypsin digestion and cyanogen bromide cleavage indicated no sequence identity with any known cytokine or chemotactic factor but revealed up to 55% sequence homology with S100, Ca2(+)-binding proteins. CP-10 appears to be the first protein of this family with a well defined function affecting cell migration, and its biological potency suggests an important role for this cytokine in cellular immune reactions.     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 2. Identification of the interacting F1 subunits by cross-linking

Interactions between oligomycin sensitivity conferring protein (OSCP) and subunits of beef heart mitochondrial F1-ATPase have been explored by cross-linking at an OSCP/F1 molar ratio close to 1 to ensure specific high-affinity binding of OSCP to F1 [see Dupuis et al. [Dupuis, A., Issartel, J.-P., Lunardi, J., Satre, M., & Vignais, P.V. (1985) Biochemistry (preceding paper in this issue)]]. Cross-links between F1 subunits and OSCP were established by means of two zero length cross-linkers, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxydihydroquinoline. The cross-linked products were separated by sodium dodecyl suflate-polyacrylamide gel electrophoresis. Coomassie blue staining revealed two cross-linked products of Mr 75 000 and 80 000 which could result from the binding of OSCP to the alpha and beta subunits of F1. Definite identification of the cross-linked products was achieved by chemical labeling with specific radiolabeled reagents and by blotting on nitrocellulose filters followed by immunocharacterization with anti-alpha, anti-beta, and anti-OSCP antibodies. OSCP was found to cross-link with the alpha and beta subunits of F1.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondria protein with an N-terminal mitochondrial targeting sequence and induces apoptosis

Apoptosis is a genetically determined cell suicide program. Mitochondria play a central role in this process and various molecules have been shown to regulate apoptosis in this organelle. In the present study, we firstly identified that protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondrial protein, which may induce apoptosis in HEK293T and HeLa cell lines. PTPIP51 transfection resulted in the externalization of phosphatidylserine (PS), activation of caspase-3, cleavage of PARP, and condensation of nuclear DNA. Further investigation revealed that PTPIP51 over-expression caused a decrease in mitochondrial membrane potential and release of cytochrome c, suggesting that it may be involved in a mitochondria/cytochrome c mediated apoptosis pathway. We also found that a putative TM domain near the N terminus of PTPIP51 is required for its targeting to mitochondria, as evidenced by the finding that deletion of the PTPIP51 TM domain prevented the protein's mitochondiral localization. Furthermore, this deletion significantly influenced the ability of PTPIP51 to induce apoptosis. Taken together, the results of the present study suggest that PTPIP51 is a mitochondrial protein with apoptosis-inducing function and that the N-terminal TM domain is required for both the correct targeting of the protein to mitochondria and its apoptotic functions.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

朱砂叶螨HSP90基因克隆及原核表达

采用RT-PCR及RACE技术克隆朱砂叶螨Tetranychus cinnabarinus的热激蛋白90(HSP90)基因, 并进行序列分析, 得到一条长2 595 bp的cDNA序列, 该序列开放阅读框（open reading frame, ORF）为2 169 bp, 编码722个氨基酸, 分子量约为83.45 kDa, 理论等电点为4.81, 3′非编码区（untranslated region, UTR）为249 bp, 5′UTR为177 bp。通过Antheprot分析发现5个HSP90家族的签名序列及胞质HSP90特征序列MEEVD。同源性分析表明, 朱砂叶螨HSP90编码区核苷酸序列和其他已知的HSP90, 尤其是节肢动物昆虫的HSP90, 具有很高的相似性。将鉴定正确的原核重组表达质粒pET43a-TcHSP90, 转化大肠杆菌Escherichia coli BL21(origami) 进行原核表达, 应用SDS-PAGE和Western blotting技术分离并检测融合蛋白, 结果表明构建的原核表达质粒可以在宿主菌中稳定、正确表达。朱砂叶螨TcHSP90基因的克隆、原核表达, 为进一步研究HSP90的性质和功能的研究提供有用的实验材料。     

Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.     

Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species

The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.     

Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Specificity of an antibody to a subunit of high-molecular-weight storage protein from wheat seed and its reaction with other cereal storage proteins (prolamins)

An antiserum to subunit 2 from the high-molecular-weight (HMW) subunits of the glutenin fraction of Triticum aestivum cv. Highbury was shown to react with related subunits from other cultivars of wheat. The reaction was measured quantitatively by laser nephelometry in polyethylene glycol phosphate-buffered saline after dissolving the HMW fraction in 0.1 M acetic acid; urea used to dissolve the HMW prolamins inhibited the reaction, in some cases at the low concentration of 0.06 M. A study of the comparative reactions of other cereal prolamins was made. D hordein, the homologous HMW protein of barley, showed less reaction, which was more inhibited by urea than the wheat subunits. Some -gliadins from the wheat cultivars Chinese Spring and Cheyenne reacted more strongly than the injected fraction and there was less inhibition by urea. A-, - and ₃ of wheat also reacted with the antiserum while a secalin of rye of M_r 40000 gave a weak reaction.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - PBS phosphate-buffered saline - PE pyridylethylated - SDS sodium dodecyl sulphate     

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.     

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-OOmethylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M_r values of 250 and 450 kDa attributed to the two subunits, values of 2.18 × 10⁵ daltons and 4.30 × 10⁵ daltons were calculated for the polypeptide part of the “light” and “heavy” subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man 3MeGal > GlcNAc GalNAc and Fuc Xyl