Estrogen-modulated estrogen receptor x Pit-1 protein complex formation and prolactin gene activation require novel protein synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.     

Post-synthetic modification of proteins

Process of postsynthetic modifications of proteins in norm are considered. Two basic groups of modification processes are singled out: 1) processes which promote appearance of derivatives for 20 basic amino acids, i.e. change in the primary structure; 2) processes which are not associated with appearance of new amino acids but are responsible mainly for the changes in the polypeptide chain conformation and size. Modification processes of amino acid variations by means of methylation, acetylation, acylation, phosphorylation, ADP-ribosylation, glycosylation, amidation, hydroxylation and metal addition are described as referred to the first group. Proteolysis reactions are characterized in detail. Their significance for formation of biologically active peptides is considered. A notion "one gene--one protein" is thought to be incompetent because formation of a number of proteins necessitates participation of tens and hundreds of genes, coding enzymes of postsynthetic modification.     

Biosynthesis of iron-sulphur clusters is a complex and highly conserved process

Iron-sulphur ([Fe-S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe-S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe-S] clusters and their insertion into various protein partners. Among the [Fe-S] cluster biosynthetic proteins are included a pyridoxal phosphate-dependent enzyme (NifS) that is involved in the activation of sulphur from l-cysteine, and a molecular scaffold protein (NifU) upon which [Fe-S] cluster precursors are formed. The formation or transfer of [Fe-S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe-S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe-S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe-S] cluster formation that were discovered in the eubacteria.     

Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria.

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.     

Developmental expression of the myelin gene MOBP in the rat nervous system

The myelin-associated/oligodendrocyte basic proteins (MOBPs) are recently discovered constituents of myelin and are small, cytoplasmic, and highly basic proteins exclusively expressed postnatally by oligodendrocytes. Due to a clustering of positively charged amino acids observed in the most abundant MOBP isoform similar to myelin basic protein (MBP) and P0, it was speculated that MOBP could function in myelin sheath compaction. The present report strongly supports this view. A direct comparison of MBP and proteolipid protein (PLP) gene expression with that of MOBP by in situ hybridization revealed a very similar regional distribution. It was found that MOBP expression was abundant in the rat CNS at postnatal day 15 (P 15) but is restricted to densely myelinated regions. In contrast to MBP and PLP, expression of MOBP was undetectable in the peripheral nervous system during the entire development. Interestingly, MOBP mRNA was localized in oligodendrocyte processes even at early postnatal stages and throughout development. MOBP showed a very specific timing of expression: in spinal cord and brain, MOBP gene expression occurred significantly later (2–3 days) than that of MBP and PLP, but slightly earlier than myelin oligodendrocyte glycoprotein gene expression. MOBP proteins appeared in spinal cord and brain stem also after MBP protein, suggesting that the MOBPs functionally act after the structural myelin proteins MBP and PLP. Our findings imply a function of MOBP during the late steps of myelin formation, presumably at the initiation of sheath compaction.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Variabilité biochimique et génétique de Sphaeroma ghigii,S. hookeri et S. serratum. Variations intraspécifiques

Electrophoresis on polyacrylamide gel of enzymes (esterases, phosphatases) and total proteins of Sphaeroma ghigii, S. hookeri and S. serratum shows the existence of important intraspecific variability. Esterases, alkaline phosphatases and major low ionic strength extractable proteins also demonstrate quantitative and qualitative differences between individuals; these differences are the expression of protein and enzyme polymorphism. Some of these variations represent sexual differences which are an expression of a biochemical sexual dimorphism that appears particularly in esterases and phosphatases. Among males, this variability leads to the delineation of several biochemical phenotypes and gives an insight into the genetic variability of populations based on the protein composition of organisms. Taking into consideration the ecological characteristics of the various species of genus Sphaeroma, this polymorphism provides a source of information on ecophysiological relations at the level of proteins and enzymes, that may lead to a better understanding of intraspecific variability.     

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.     

近暗散白蚁β-葡糖苷酶基因RpBg7的克隆及其在毕赤酵母中的表达

【目的】白蚁是自然界中利用木质纤维素能力很强的生物,是纤维素酶的天然资源库。本研究旨在挖掘新来源的纤维素酶基因,为生物质能源的高效利用提供新的天然酶。【方法】根据前期蛋白质组测序的结果,利用PCR结合RACE克隆了近暗散白蚁Reticulitermes perilucifugus β-葡糖苷酶7(β-glucosidase 7)基因RpBg7 cDNA全长序列;通过生物信息学软件分析了RpBg7的序列;用表达载体pPICZαA在毕赤酵母Pichia pastoris X-33中表达RpBg7蛋白,并用4-硝基苯基-β-d-吡喃葡萄糖苷(4-nitrophenyl β-d-glucopyranoside, 4pNPG)为底物检测了表达的RpBg7蛋白的酶活性。【结果】获得了近暗散白蚁的一个内源性β-葡糖苷酶7基因RpBg7(GenBank登录号: MN944395),其开放阅读框长1 485 bp,编码495个氨基酸残基。RpBg7蛋白预测分子量为57 kD,属于糖苷水解酶1(glycosidehydrolase 1, GH1)家族,具有保守的碱性氨基酸残基Glu187和Glu394。通过毕赤酵母表达系统成功表达RpBg7蛋白。酶活性分析结果表明,毕赤酵母胞外分泌蛋白粗酶液和胞内蛋白粗酶液中RpBg7酶活性分别为4.43和7.47 U/mL。【结论】克隆并利用毕赤酵母表达了近暗散白蚁的GH1家族的一个β-葡糖苷酶7基因RpBg7,为后期纤维素酶的改造和应用提供了条件。     

Sexual dimorphism of rat liver nuclear proteins: regulatory role of growth hormone

Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n=9) and female (n=5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n=5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p<0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus.     

The genetic basis of constitutive and herbivore-induced ESP-independent nitrile formation in Arabidopsis

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system.     

CD154 variants associated with hyper-IgM syndrome can form oligomers and trigger CD40-mediated signals

X-linked hyper-IgM syndrome is a rare immunodeficiency disorder resulting from mutations in the gene encoding the CD40 ligand (CD154) molecule. These mutations are very heterogeneous, ranging from a single point mutation to a large deletion in the open reading frame. To investigate the molecular mechanisms that are responsible for the functional defect of these mutants, we examined the biochemical properties of 14 hyper-IgM-related CD154 mutant proteins produced by transient expression in COS7 cells. We show that deletion mutants lacking a significant portion of the tumor necrosis factor homologous domain cannot be stably produced. In contrast, point mutants can be detected as oligomers. Surprisingly, gene products of two point mutants, Thr-211 --> Asp and Met-36 --> Arg, can bind to the receptor, CD40. For Thr-211 --> Asp variant, it is comparable to the wild-type protein in its surface expression level, biochemical structure, and functional activities. Thus, it appears that this mutation is a polymorphism of CD154 gene. For Met-36 --> Arg variant, although it is interactive with CD40, it has a much lower surface expression level than wild-type protein. We propose that Met-36 --> Arg mutant represents a prototype of a defective CD154 family whose low cell surface expression of intrinsically active protein is simply insufficient to trigger productive signals through CD40.     

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

Abstract The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.