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PubMed1992

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.

Amino Acid Sequence↗

PubMed1991

Renal function, protein excretion and pathology of Balkan endemic nephropathy. I. Renal function.

Renal function studies were performed on 59 patients who had the clinical criteria for Balkan endemic nephropathy (BEN). They were divided into three groups according to DTPA clearance (DTPA). Group 1, 11 individuals, had a mean age of 41.6 years and DTPA greater than 100 ml/min. Group 2, 20 persons, had a mean age of 49 years and DTPA of 60 to 100 ml/min. Group 3 was made up of 28 people with a DTPA less than 60 ml/min and an average age of 50.4 years. No distinguishing specific or characteristic symptoms of BEN were found in any of the three groups. Anemia was not found to be an early indicator when compared to other forms of progressive renal disease. Proteinuria was minimal and intermittent in all three groups. Maximum concentrating ability was significantly reduced only in the third group. These features do not allow the clinical differentiation of BEN from other chronic progress tubulointerstitial nephropathies. The geographic isolation and familial nature of the disease, associated with minimal proteinuria make BEN a unique entity. Kidney size by ultrasound was decreased in all three groups, suggesting that this may be another early and characteristic feature to BEN.

Adult↗

PubMed1985

The T4 mot protein functions as part of a pre-replicative DNA-protein complex.

Middle-mode RNA synthesis in T4-infected cells takes place before replication of phage DNA commences. What distinguishes it from early-mode RNA synthesis is that initiation of middle RNA depends on T4-coded proteins, in particular on the mot gene product. mot protein is localized in a DNA-protein complex which forms during the first few minutes of infection. All of the cell's mot protein is bound in this complex, and it continues to be bound long after the synthesis of mot protein has stopped. When we infect Escherichia coli with T4 carrying a temperature-sensitive mutation in the mot gene, we find a correlation between the physiology of this mot mutant and the amount of mot protein bound in the DNA-protein complex. Although there is some host RNA polymerase in the complex, mot protein does not seem to bind to this enzyme. Two other T4-coded proteins, of molecular weights 17,600 and 15,000, are also found in the pre-replicative DNA-protein complex. One of these, p17,600, is coded for by a 750-base pair region located between genes 39 and 56; p17,600 appears to be the recently described motB gene product. The other protein, p15,000, is not an RNA polymerase-binding protein; it is characterized by its strong binding to the DNA-protein complex.

DNA Replication↗

PubMedDate not supplied

Molecular aspects of fatty acid transport: mutations in the IYTSGTTGXPK motif impair fatty acid transport protein function.

The murine fatty acid transport protein (FATP) facilitates uptake of long chain fatty acids (LCFAs) when expressed in mammalian cells. FATP's sequence contains a highly conserved motif, IYTSGTTGXPK, also found in a number of proteins known to interact with ATP. To explore the role of this motif, we independently mutated the central serine (serine 250) and threonine (threonine 252) residues in this motif and assessed the effects of these mutations on FATP function. When expressed in fibroblasts, the FATP mutants demonstrated impaired LCFA import and impaired binding of [alpha-32P]8-azido-ATP (azido-ATP) compared with wild-type FATP. These results suggest that serine 250 and threonine 252 are critical for FATP function and that the mechanism of action of FATP involves nucleotide binding which is dependent on these residues.

3T3 Cells↗

PubMed1996

Mechanisms of Bcl-2 family protein function and dysfunction in health and disease.

The Bcl-2 protein blocks a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis. The gene encoding this protein was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in B-cell lymphomas, where it contributes to neoplastic cell expansion by preventing cell turnover due to programmed cell death. Overexpression of BCL-2 also occurs in many other types of human tumors, including cancers of the prostate, colon, and lung, and has been associated with chemoresistance and radioresistance in some types of malignancy. Conversely, expression of BCL-2 is frequently reduced in the circulating lymphocytes of persons infected with Human Immunodeficiency Virus (HIV), which are prone to apoptotic cell death. Since the discovery of Bcl-2 a decade ago, several other cellular and viral genes encoding homologous proteins have been identified, some of which suppress cell death akin to Bcl-2 (Bcl-XL, Mcl-1, A1/Bfl-1, Nr13, Ced-9, BHRF-1) and others which promote apoptosis (Bax, Bcl-Xs, Bak, Bik, Bad). Several of these Bcl-2 family proteins are capable of physically interacting with each other through a complex network of homo- and heterodimers. The expression of some of these other BCL-2 family genes becomes altered in human cancers, as well as in the setting of ischemia and some other pathological conditions, suggesting a potentially important role for these Bcl-2 homologs in human diseases characterized by either insufficient or excessive cell death. Despite intensive investigation, the mechanisms by which Bcl-2 and its homologs control cell life and death largely remain enigmatic. Knowledge about the specific domains in Bcl-2 family proteins that are required for interactions with other proteins and for function however is beginning to provide insights into the molecular mechanisms through which these proteins regulate the programmed cell death pathway in normalcy and disease.

Animals↗

PubMed1991

G protein function in the ischaemic myocardium.

The activity of adenylyl cyclase (AC) is controlled by its interaction with receptor-regulated G proteins. The efficiency to form cyclic AMP is strongly influenced by the amount, the subspecies and function of these regulatory proteins. An impairment of AC function has been shown to occur in sarcolemmal preparations (SL) of hearts exposed to either local or global ischaemia. To examine the contribution of G protein function to this phenomenon, cholera toxin (CT)-catalysed ADP-ribosylation of Gs and pertussis toxin (PT)-catalysed ADP-ribosylation of G proteins have been investigated in SL of porcine hearts exposed to global ischaemia for 15-45 min. ADP-ribosylation by CT of an approximately 45 kDa polypeptide was 0.46 +/- 0.06 and ADP-ribosylation by PT of three 39-41 kDa polypeptides was 4.77 +/- 0.77 pmol mg-1 protein in SL of non-ischaemic myocardium. Whereas no change was observed in CT-catalyzed ribosylation after 30 min of ischaemia, there was a reduction in PT-catalyzed ADP-ribosylation to 3.7 +/- 0.35 pmol mg-1 protein after 30 min of ischaemia. Prolongation of ischaemia to 45 min did not reduce further ADP-ribosylation capacity. Quantitative immunoblotting of PT-sensitive G proteins suggests that the diminution of ADP-ribosylation occurred because of a loss of alpha-subunits of G0, Gi-1, and Gi-2 from sarcolemmal membranes.

Adenosine Diphosphate Ribose↗

PubMed1990

Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria.

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.

Bacteria↗

PubMed1997

Assessment of cholesteryl ester transfer protein function in lipoprotein mixtures by 1H NMR spectroscopy.

Studies of cholesteryl ester transfer protein (CETP) function in lipoprotein mixtures pose many difficulties by conventional biochemical methods. For instance, studies on the effects of CETP on the composition of apolipoprotein B containing lipoproteins (very low and low density lipoproteins) in lipoprotein mixtures are tedious due to repeated ultracentrifugational isolations and have thus rarely been performed. Here we present a new 1H NMR spectroscopy technique to assess the CETP function in lipoprotein mixtures. This technique does not require repeated physical isolations of the lipoprotein particles but uses mathematical separation of the fractions on the basis of biochemical prior knowledge based lineshape fitting analysis of specific lipid resonances in the 1H NMR spectra. The lipoproteins are separated according to their size related chemical shift which allows for distinct quantification between very low and low density lipoproteins, the two major apolipoprotein B containing fractions. The methodological basis of the technique is discussed here together with a demonstration that this kind of approach allows dynamic follow up of the lipid transfer reactions in complex lipoprotein and CETP mixtures. The results revealed a consistent behaviour which corroborated the recent findings suggesting that the neutral lipid mass transfer among lipoproteins is not an equimolar heteroexchange.

Apolipoproteins↗

PubMed1998

Cysteine string protein functions directly in regulated exocytosis.

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.

Animals↗

PubMed1992

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.

Cell Nucleus↗

PubMed1991

Frequencies of codons in histones, tubulins and fibrinogen: bias due to interference between transcription signals and protein function.

The distribution of codons was studied in 65 proteins: 48 histones, 14 tubulins, and three fibrinogens, With the methodology used, (1) we confirmed that the preterminator state of a codon has no detectable effect on codon bias. (2) The well-known effect of CG suppression was visible. We also found that (3) some codons which are very rare, are equal to parts of known transcription signals. Thus, we advanced that to avoid signal interference, the use of these codons is suppressed when a synonymous codon is available. In addition we found that in the whole series of codons, transcription signals are less frequent than in a random sequence of equal composition. Finally we observed (4) that tryptophan is absent in histones. This absence was related not to the TGG codon itself, but to characteristics of the amino acid. We conclude that the functional constraints of a protein can influence, at least for synonymous codon usage, the evolution of its own coding sequence.

Animals↗

PubMed1990

A human ubiquitin carboxyl extension protein functions in yeast.

The ability of the human ubiquitin carboxyl extension protein (HUBCEP80) to functionally replace its yeast homolog was determined in a ubi3 mutant of Saccharomyces cerevisiae. Expression of HUBCEP80 in ubi3 mutants resulted in processing of the fusion protein to produce free ubiquitin and extension protein, the latter of which localized specifically with the 40 S ribosomal subunit. Furthermore, expression of the human fusion protein completely alleviated the phenotypic deficiencies found in ubi3 mutants, including slow growth, abnormal ribosomal RNA processing, and correspondingly low levels of 40 S ribosomal subunits. Finally, expression of the extension protein alone was much less efficient in complementing the ubi3 mutant phenotype as compared with expression of the normal ubiquitin-fused extension protein. In the latter case, cells were found to contain at least 5-fold more extension protein, suggesting that ubiquitin either increased translational efficiency of the HUBCEP80 transcript or increased the stability of the processed extension protein.

Chromosome Deletion↗

PubMed1993

Modularity of protein function: chimeric interleukin 1 beta s containing specific protease inhibitor loops retain function of both molecules.

Although it is widely recognized that many proteins contain discrete functional domains, it is less certain whether smaller, less obviously discrete, units of structure will retain their specific function when transplanted into a different context. The observation that the potent inflammatory cytokine human interleukin 1 beta has the same overall structure as soybean trypsin inhibitor (STI) (Kunitz) prompted us to replace a tight turn in the cytokine sequence with the large loop in soybean trypsin inhibitor that binds to the active site of trypsin. Wild-type interleukin 1 beta (IL-1 beta) is highly resistant to proteolysis, but the chimeric STI/IL is specifically cleaved by trypsin, apparently in the inserted loop. Other chimeric interleukins have also been constructed, by replacing the same tight turn with inhibitory loops from other protein protease inhibitors: turkey ovomucoid inhibitor (TOI), a chymotrypsin inhibitor, and alpha 1-antitrypsin (AT), an elastase inhibitor. Although these loops come from proteins not related structurally to interleukin 1, they confer specific protease sensitivity or inhibition on the chimeric cytokine. The cytokine properties of these chimeric interleukins have also been evaluated. The chimeras formed from human IL-1 beta and all inhibitory loops tested bind to the interleukin 1 receptor with reasonable affinity. The typical cellular effects of IL-1, however, are not observed with all the recombinant proteins, thus confirming that receptor binding and signal transduction can be uncoupled. When these results are taken together with the results of site-directed mutagenesis of IL-1, reported in this paper and elsewhere, they allow the receptor and intracellular transduction sites on the protein to be mapped in detail.

Amino Acid Sequence↗

PubMed1999

Mutational analysis of cysteine-string protein function in insulin exocytosis.

Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.

Cells, Cultured↗

PubMed1996

Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality.

Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Both FGF-2 peptides also induced functional modifications of G-proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP-ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMedDate not supplied

Coagulation protein function. IV. Effect of acetaldehyde upon factor X and factor Xa, the proteins at the gateway to the common coagulation pathway.

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russell's viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohol's primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Acetaldehyde↗

PubMed1991

Human genome protein function database.

A database which focuses on the normal functions of the currently-known protein products of the Human Genome was constructed. Information is stored as text, figures, tables, and diagrams. The program contains built-in functions to modify, update, categorize, hypertext, search, create reports, and establish links to other databases. The semi-automated categorization feature of the database program was used to classify these proteins in terms of biomedical functions.

Databases, Factual↗

PubMed1996

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.

Actin Capping Proteins↗