An essential proline in lambda repressor is required for resistance to intracellular proteolysis

Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, J.F., & Sauer, R.T. (1990) Proteins: Struct., Funct., Genet. (in press)]. To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases. The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli

To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.     

Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.     

Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli . The P1 promoter of topA has previously been shown to be a σ³²-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10-fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.     

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.     

Defect in general priming conferred by linker region mutants of Escherichia coli dnaB.

The dnaB gene of Escherichia coli encodes a bifunctional primase accessory protein/helicase necessary for chromosomal replication. Monomers of DnaB comprise two trypsin-resistant domains connected by a 45-amino-acid linker. To investigate the role of the linker in the structure and function of DnaB, we have purified and characterized three DnaB mutant proteins having single amino acid substitutions in the linker. We find that the mutant proteins retain the two-domain structure and assemble into hexamers that may be less stable than hexamers formed by wild-type DnaB. These mutant hexamers have hydrodynamic properties slightly different from those of the wild type, suggestive of a more open structure. The mutant proteins had reduced or absent ability to stimulate primase and also exhibited slight alterations in ATPase activity compared with the wild type. We conclude that the linker region promotes primase-DnaB interaction, but this effect may be indirect. We propose a model involving repositioning of N-terminal domains to explain the properties of the mutant proteins.     

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.     

Single tryptophanyl substitutions affect the structure of apomyoglobin

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions A-5 (W7) and A-12 (W14) in the N-terminal region (A helix) of the protein molecule. To determine the contribution of each tryptophanyl residue to the structure and stability of myoglobin, recombinant proteins with single indole residue, i.e., W7 or W14, were obtained by site-directed mutagenesis. The mutant proteins, expressed in Escherichia coli, were found correctly folded, the far ultraviolet circular dichroism of both mutants as well as the Soret absorption being superimposed to that of wild type protein. The removal of the prosthetic group from mutant proteins determined a loss of helical content much larger than that observed in the case of wild type myoglobin. These results suggest that tryptophanyl residues can play a crucial role on globin folding and structure.     

Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation.

Because of the highly conserved pattern of expression of the eucaryotic heat shock genes hsp70 and hsp84 or their cognates during sporulation in Saccharomyces cerevisiae and development in higher organisms, the role of the Escherichia coli homologs dnaK and htpG was examined during the response to starvation. The htpG deletion mutant was found to be similar to its wild-type parent in its ability to survive starvation for essential nutrients and to induce proteins specific to starvation conditions. The dnaK103 mutant, however, was highly susceptible to killing by starvation for carbon and, to a lesser extent, for nitrogen and phosphate. Analysis of proteins induced under starvation conditions on two-dimensional gels showed that the dnaK103 mutant was defective for the synthesis of some proteins induced in wild-type cells by carbon starvation and of some proteins induced under all starvation conditions, including the stationary phase in wild-type cells. In addition, unique proteins were synthesized in the dnaK103 mutant in response to starvation. Although the synthesis of some proteins under glucose starvation control was drastically affected by the dnaK103 mutation, the synthesis of proteins specifically induced by nitrogen starvation was essentially unaffected. Similarly, the dnaK103 mutant was able to grow, utilizing glutamine or arginine as a source of nitrogen, at a rate approximate to that of the wild-type parent, but it inefficiently utilized glycerol or maltose as carbon sources. Several differences between the protein synthetic pattern of the dnaK103 mutant and the wild type were observed after phosphate starvation, but these did not result in a decreased ability to survive phosphate starvation, compared with nitrogen starvation.     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.     

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.     

GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes

Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.     

Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).