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At least 973 records · Page 54Linked to original sources
PubMed2001

Proteolytic activation of SREBPs during adipocyte differentiation.

A member of sterol regulatory element-binding protein (SREBP) family, SREBP-1, is a key regulator of adipocyte differentiation. Expression of the SREBP-1 gene is induced during adipocyte differentiation, but proteolytic activation of the synthesized precursor form of SREBP-1 has not been well analyzed. The proteolytic processing of SREBPs is severely suppressed in sterol loaded culture cells. Here we report that a splicing isoform, SREBP-1a, is predominantly expressed in 3T3-L1 preadipocytes and adipocytes, and that the nuclear active form of SREBP-1 protein increases in adipocyte differentiation. We further show that the amount of nuclear SREBP-2 protein also increases despite no increase in SREBP-2 mRNA, suggesting that proteolytic cleavage of SREBPs is induced in lipid loaded adipocytes. Northern blot analyses reveal that mRNA levels for SREBP cleavage-activating protein (SCAP), Site-1 protease (S1P), and Site-2 protease (S2P), which participate in the proteolytic processing of SREBPs, are relatively unaffected in adipogenesis. These results demonstrate that SREBP-2 appears to promote adipocyte differentiation as well as SREBP-1 and that the proteolytic activation of SREBPs may be induced by an as-yet unidentified mechanism in lipid loaded adipocytes.

3T3 Cells↗
PubMed2001

Nitric oxide inhibits selectively the 17beta-estradiol-induced gene expression without affecting nongenomic events in HeLa cells.

17beta-Estradiol (E2) induces genomic (i.e., pC3-luciferase promoter-reporter construct expression) and nongenomic (i.e., DNA synthesis and IP(3) production) effects in HeLa cells only after transient transfection with the human estrogen receptor alpha (ERalpha) reporter plasmid. Here the effect of nitric oxide (NO) on both E2-induced effects in transiently transfected HeLa cells is reported. Remarkably, the E2-dependent gene transcription is inhibited dose-dependently by NO. By contrast, DNA synthesis and IP(3) production, representing nongenomic E2-dependent effects, are unaffected by NO. The selective NO action on E2-induced functions may be related to NO-mediated chemical modification(s) of the Cys residues present in the DNA recognition domain of ERalpha impairing DNA binding.

Aspartic Acid Endopeptidases↗
PubMed2000

Trypanosoma cruzi: a putative vacuolar ATP synthase subunit and a CAAX prenyl protease-encoding gene, as examples of gene identification in genome projects.

An international genome program has been initiated to increase the knowledge about the Trypanosoma cruzi genome and thereby find effective tools to treat Chagas' disease. We here report the molecular characterization of two novel genes found in the course of this project. Two of the open reading frames (ORF) identified in the sequencing of the third smallest chromosome of the CL Brener strain of T. cruzi were selected for further molecular characterization due to their similarity to genes with interesting functions in other organisms and their potential as targets to combat the parasite. The first ORF (402 bp) showed homology to a 14-kDa vacuolar ATP synthase subunit F from a variety of organisms, such as yeast, rat, bovine, human, and a number of prokaryotes. The second ORF (1188 bp) resembled a CAAX prenyl protease-encoding gene, identified in different organisms, including Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana, as well as several prokaryotes. RT-PCR from T. cruzi total epimastigote RNA allowed us to isolate the complete transcripts of these genes. Furthermore, screening of an available normalized cDNA library derived from the same stage of the parasite confirmed that both genes are expressed at least in the epimastigote stage of T. cruzi. Comparison of the putative T. cruzi proteins to their counterparts in other organisms revealed significant protein sequence conservation over large evolutionary distances. Computer analysis revealed the presence of several motifs in both proteins, possibly related to the regulation and localization of these proteins in the parasite.

ATP Synthetase Complexes↗
PubMed1999

Identification and chromosomal location of two human genes encoding enzymes potentially involved in proteolytic maturation of farnesylated proteins.

Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library. These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases. Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes. Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas. On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins.

Amino Acid Sequence↗
PubMed2000

Cloning of PC3B, a novel member of the PC3/BTG/TOB family of growth inhibitory genes, highly expressed in the olfactory epithelium.

We identified in the EST database murine and human sequences similar, but not identical, to the members of the PC3/BTG/TOB family of cell cycle inhibitors. A conserved domain (aa 50-68) of the PC3 protein, the prototype member of the family, was used as a query. That domain has been shown by us to be necessary for the antiproliferative activity of PC3. A murine EST clone and a highly homologous human EST clone, containing the entire ORF, were chosen for sequencing. Comparison to databases and a phylogenetic tree analysis indicated that these EST clones are the mouse and human homologues of a gene that represents a novel member of the PC3/BTG/TOB family. This gene, named PC3B, is endowed with marked antiproliferative activity, being able to induce G(1) arrest, and is highly expressed in testis, in oocyte, and in preimplantation embryos. Analysis of its expression during murine development indicated a specific localization in the olfactory epithelium at midgestation, suggesting that PC3B might be involved in the differentiation of this neuronal structure. Human PC3B mapped to chromosome 11q23, as indicated by radiation hybrid analysis.

Amino Acid Sequence↗
PubMed1997

Phospholipase C isoforms delta 1 and delta 3 from human fibroblasts. High-yield expression in Escherichia coli, simple purification, and properties.

Phospholipase C isoforms delta 1 and delta 3 (PLC delta 1 and delta 3) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts. The enzymes were also expressed with the sequence Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as "-H6K2," attached to their amino termini. PLC delta 1, PLC delta 1-H6K2, PLC delta 3, PLC delta 3-H6K2 all expressed in highly active form. The H6K2-bearing isoforms were each purified to homogeneity in a single step, with yields of 90-100%, using agarose-iminodiacetic acid-Ni columns and imidazole buffer as eluting agent. Yields in terms of activity increased as the temperature of expression was decreased. Expression at 16 degrees C for 72 h yielded 33 mg of pure PLC delta 1-H6K2 and 13 mg of pure PLC delta 3-H6K2 per liter of culture. Removal of H6K2 from both isoforms with Kex2 protease resulted in little or no loss of activity. Expression of PLC isoforms bearing -H6K2 at the amino terminus resulted in about 12 times more activity than expression of the isoforms lacking -H6K2. PLC delta 3 is much less stable than PLC delta1. Successful purification and storage of PLC delta 3 depends on a suitable stabilizing medium. Both isoforms require 0.3 microM calcium ion for half-maximum activity. The specific activities of the isoforms expressed with and without -H6K2 are the same, as are their calcium saturation curves.

Amino Acid Sequence↗
PubMed1998

Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae.

Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor from S. cerevisiae (T. Kjeldsen et al., Biotechnol. Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing either in vivo Kex2 endoprotease maturation of the fusion protein during secretion or in vitro maturation of the purified fusion protein with a suitable enzyme.

Alcaligenes↗
PubMed1995

Sequence of the M28 dsRNA: preprotoxin is processed to an alpha/beta heterodimeric protein toxin.

The killer and immunity phenotypes of K28 killer strains of Saccharomyces cerevisiae are determined by the 1.75-kb M28 dsRNA virus. In the plus strand, M28p, the K28 preprotoxin gene, comprises bases 13-1047 and is followed, after an additional 85 bases, by a 63-bp poly(A) sequence and a 553-base 3'-sequence. This 3'-sequence contains two potential stem-loop structures predicted to bind the L-A encoded cap-pol protein, initiating encapsidation; high-level expression results in curing of M1 dsRNA. Expression of M28p confers the complete K28 killer and immunity phenotype on a cell lacking M28 dsRNA. K28 toxin is a disulfide-bonded heterodimer of alpha (10.5 kDa) and beta (11 kDa) components whose N-termini correspond to M28p residues 50-61 and 246-257, respectively. alpha is preceded by a potentially redundant pair of secretion signal peptides; deletion of the first reduces toxin secretion by 75%. While M28p bears no sequence similarity to M1p, the K1 preprotoxin, the predicted patterns of processing by glycosylation and cleavage are remarkably similar. The beta N- and C-termini are probably processed by Kex2p and Kex1p, respectively; the mechanism of cleavage at the less typical sites bounding the alpha component is under investigation. While a kex2 delta mutation prevents toxin secretion, secreted toxin retains 20% activity in a kex1 delta mutant. Neither mutation affects immunity.

Amino Acid Sequence↗
PubMed1995

The hemagglutinin cleavability of a virulent avian influenza virus by subtilisin-like endoproteases is influenced by the amino acid immediately downstream of the cleavage site.

Many viral membrane glycoproteins are post-translationally processed by intracellular endoproteases such as subtilisin-like proteases. These proteases recognize a cleavage site sequence comprising basic amino acids positioned upstream of the cleavage site of the viral proteins. Here, we mutated the glycine residue immediately downstream of the cleavage site (P1) of hemagglutinin (HA) from a virulent avian influenza virus, A/turkey/Ontario/7732/66 (H5N9) (R-R-R-K-K-R/G), to examine the effect of this mutation on its clevability. Substitution of Gly with Ile, Leu, Val, or Pro, but not Ala, Asp, Phe, His, Ser, or Thr, resulted in substantial reduction of HA cleavage by endogenous endoproteases in CV-1 cells and by vaccinia-expressed PC6 and, albeit to a lesser extent, furin. We conclude that HA cleavage by subtilisin-like proteases is influenced by the downstream P1 amino acid in the absence of upstream cleavage site sequence alterations.

Amino Acid Sequence↗
PubMed1996

Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA.

A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pDl) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5' end of the transcript, the rapid amplification of cDNA end (5'-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.

Algal Proteins↗
PubMed1991

In-vitro processing of yeast alpha-factor leader fusion proteins using a soluble yscF (Kex2) variant.

The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the alpha-factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscFs). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for alpha-factor leader-hIGF1 and alpha-factor leader-hirudin fusion proteins.

Colorimetry↗
PubMed1992

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant.

In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.

Amino Acid Sequence↗
PubMed1990

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes.

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

Amino Acid Sequence↗
PubMed1999

A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs).

A missing component in the experimental analysis of cell signaling by extracellular lysophospholipids such as lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) has been cloned receptors. Through studies on the developing brain, the first such receptor gene (referred to as vzg-1) was identified, representing a member of the G-protein coupled receptor (GPCR) super family (1). Here we review the neurobiological approach that led to both its cloning and identification as a receptor for LPA, along with related expression data. Summarized sequence and genomic structure analyses indicate that this first, functionally identified receptor is encoded by a member of a growing gene family that divides into at least two subgroups: genes most homologous to the high-affinity LPA receptor encoded by vzg-1, and those more homologous to an orphan receptor gene edg-1 that has recently been identified as a S1P receptor. A provisional nomenclature is proposed, based on published functional ligand actions, amino acid composition and genomic structure whereby the receptors encoded by these genes are referred to as lysophospholipid (LP) receptors, with subgroups distinguished by letter and number subscripts (e.g., LPA1 for Vzg-1, and LPB1 for Edg-1). Presented expression data support the recently published work indicating that members of the LPB1 subgroup are receptors for the structurally-related molecule, S1P. The availability of cloned LP receptors will enhance the analysis of the many documented LP effects, while their prominent expression in the nervous system indicates significant but as yet unknown roles in development, normal function, and neuropathology.

Base Sequence↗
PubMed2004

Biological functions and signaling of a transmembrane semaphorin, CD100/Sema4D.

The semaphorin proteins were identified originally as axonal guidance factors functioning during neuronal development. In addition to this function, several semaphorins play diverse roles outside the nervous system. The class 4 semaphorin CD100/Sema4D, which utilizes plexin-B1 and CD72 as receptors, exerts important biological effects on a variety of cells, including the neuronal, epithelial and immune cells. Here, we review recent advances exploring the molecular mechanisms governing the biological functions of CD100/Sema4D.

Animals↗
PubMed2004

Protein structure to function: insights from computation.

Computation plays an important role in functional genomics. THEMATICS is a computational method that predicts chemical and electrostatic properties of residues in enzymes and utilizes information contained in those predictions to identify active sites. The only input required is the three-dimensional structure of the query protein. The identification of residues involved in catalysis and in recognition is discussed. The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes. In addition, Kex2 is specific for dibasic sites and THEMATICS finds the recognition residues for both the S1 and S2 sites of Kex2. In contrast, no such recognition sites are found for the non-specific enzyme subtilisin. The ability to identify sites that govern recognition opens the door to better understanding of specificity and to the design of highly specific inhibitors.

Binding Sites↗