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M Inouye

Publications and source records attributed to M Inouye.

At least 73 records · Page 4Linked to original sources
PubMed1999

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Amino Acid Sequence↗
PubMed1999

Characterization of Escherichia coli cspE, whose product negatively regulates transcription of cspA, the gene for the major cold shock protein.

Escherichia coli contains nine members of the CspA protein family from CspA to Cspl. To elucidate the cellular function of CspE, we constructed a delta cspE strain. CspE is highly produced at 37 degrees C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary-phase cells into the fresh medium at 37 degrees C. This is consistent with the delta cspE phenotype of the longer growth lag period after dilution. The protein synthesis patterns of the delta cspE strain and the wild-type strain were compared using two-dimensional gel electrophoresis. In the delta cspE strain, the synthesis of a number of proteins at 37 degrees C was found to be altered and cspA was derepressed. The derepression of cspA in the delta cspE strain was at the level of transcription in a promoter-independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA. In a cell-free protein synthesis system using S-30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37 degrees C, probably by interacting with the transcription elongation complex at the cspA cold box region.

Bacterial Proteins↗
PubMed1999

Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli.

The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity. This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature. The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified. As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX). In the present study, we show that these proteins are able to bind preferentially to specific RNA/single-stranded DNA sequences. The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT-rich regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the range 0.23-0.9 x 10(-6) M, while CspC has 10-fold lower binding affinity. Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus. This motif is the putative CspE-mediated transcription pause recognition site in a 5'-untranslated region of the cspA mRNA.

Bacterial Proteins↗
PubMed1999

Histidine kinases: diversity of domain organization.

Histidine kinases play a major role in signal transduction in prokaryotes for the cellular adaptation to environmental conditions and stresses. Recent progress in the three-dimensional structure determination of two representative members of histidine kinases, EnvZ (class I) and CheA (class II), has revealed common structural features, as well as a kinase catalytic motif topologically similar to those of the ATP-binding domains of a few ATPases. They have also disclosed that there are significant differences in domain organization between class I and II histidine kinases, possibly reflecting their distinct locations, functions and regulatory mechanisms. In spite of this diversity, both class I and II histidine kinases use similar four-helix bundle motifs to relay phosphoryl groups from ATP to regulatory domains of response regulators. The previously known so-called transmitter domain of histidine kinase is further dissected into two domains: a CA (Catalytic ATP-binding) domain and a DHp (Dimerization Histidine phosphotransfer) domain for class I, or a CA domain and an HPt (Histidine-containing Phosphotransfer) domain for class II histidine kinases. From a comparative analysis of the CA domains of EnvZ, CheA and their ATPase homologues, the core elements of the CA domain have been derived. The apparent resemblance between DHp and HPt domains is only superficial, and significant differences between them are discussed.

Adenosine Triphosphate↗
PubMed1999

A sequence downstream of the initiation codon is essential for cold shock induction of cspB of Escherichia coli.

Cold shock induction of cspB has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of cspB at low temperature also requires the translational cis-acting element called the downstream box (DB). Full induction of cspB at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.

Bacterial Proteins↗
PubMed1999

Mutation analysis of the 5' untranslated region of the cold shock cspA mRNA of Escherichia coli.

The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C. However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase. Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase. In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.

5' Untranslated Regions↗
PubMed1999

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock.

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).

5' Untranslated Regions↗
PubMed1999

CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis.

CspA, CspB, and CspG, the major cold shock proteins of Escherichia coli, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37 degrees C in the presence of kanamycin (100 microgram/ml) or chloramphenicol (200 microgram/ml). After 10 min of incubation with the antibiotics at 37 degrees C, cells were cold shocked at 15 degrees C and labeled with [35S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of cspA and cspB mRNAs for the first hour at 15 degrees C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.

Amino Acids↗
PubMed1999

Expression and secretion of proteins in E. coli.

This review outlines approaches to the cloning and expression of proteins in Escherichia coli. The expression vectors described here (pIN-III derivatives) utilize the strong lipoprotein promoter, which is controlled by the lac-UV5 promoter-operator. These vectors provide the means for targeting a protein to any of the four subcellular compartments of the bacterial cell: cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Of particular importance is that secretion of proteins into the E. coli periplasm (using the OmpA signal peptide) is applicable for the production of both prokaryotic and eukaryotic proteins thereby enhancing protein activity and stability.

Amino Acid Sequence↗
PubMed1999

Histologic changes after semicircular canal occlusion in guinea pigs.

HYPOTHESIS: Histologic changes occurring after varying degrees of surgical trauma to the inner ear in guinea pigs can reveal the mechanism of hearing preservation/loss. BACKGROUND: Surgical approaches to the inner ear that allow for hearing preservation have gained increasing acceptance in neurotologic surgery. The mechanisms responsible for hearing preservation and hearing loss after partial labyrinthectomy are as yet poorly understood. METHODS: Ten animals underwent semicircular canal occlusion, suctioning of perilymph, ampullectomy, or wide vestibulotomy. Tone-burst auditory brain stem response (ABR) thresholds were performed at weekly intervals after surgery. After 4 weeks, temporal bone specimens were processed to obtain 10-jim sections from plastic-embedded ears. The histologic findings were correlated with the initial and final ABR thresholds. RESULTS: After surgical occlusion of one or more semicircular canals, ABR thresholds were preserved, as the authors reported previously. Suctioning of inner ear fluid led to transient loss of thresholds with recovery. Ampullectomy produced dichotomous results, with some subjects preserving auditory function and others losing auditory function. Wide vestibulotomy resulted in permanent loss of auditory function in most cases. Histologically, there was intraluminal fibrosis and inflammation near the site of surgical entry. Most specimens showed normal cochlear architecture and hair cell counts, irrespective of the degree of hearing loss. Vestibular hair cells were also well preserved, even when they were close to the site of surgical injury. CONCLUSIONS: These findings suggest that electromechanical changes, rather than cell death, are responsible for changes in auditory and vestibular function after partial labyrinthectomy.

Animals↗
PubMed1998

The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution.

We report here the crystallographic structure determination of an autoprocessed (Ser221Cys)-subtilisin E-propeptide complex at 2.0 A resolution. The subtilisin domain sequence has a single substitution (Ser221Cys) which has been shown to block the maturation process prior to degradation of the propeptide domain (77 residues) that acts as an intramolecular chaperon. This mutation, however, did not prevent the enzyme from cleaving its propeptide domain with a 60-80% efficiency. The current determination is the first example of a subtilisin E-propeptide complex which has been autoprocessed. A previous structure determination of a BPN'-prosegment complex has been reported in which the subtilisin domain was extensively mutated and a calcium binding loop was deleted. Further, in this earlier determination, the complex was formed by the addition of separately expressed propeptide domain. The structure determination reported here provides additional information about the nature of the interaction between the subtilisin and propeptide domains in this complex.

Amino Acid Sequence↗
PubMed1998

NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ.

Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.

Amino Acid Sequence↗
PubMed1998

Levels of lipid peroxidation product and glycated hemoglobin A1c in the erythrocytes of diabetic patients.

In diabetes, the glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether or not the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). There is some evidence, however, that glycation itself may induce the formation of OFRs. OFRs could cause oxidative damage to endogenous molecules. We examined the relationship between the levels of lipid peroxidation and the levels of glycated hemoglobin A1c (GHbA1c) in erythrocytes of diabetic and healthy subjects. Lipid peroxidation was assessed in erythrocyte membrane lipids by monitoring peak height ratios of conjugated linoleic acid (CLA), one of the products of lipid peroxidation, to linoleic acid (LA) using gas chromatography-mass spectrometry (GC/MS). CLA is a collective term used to designate a mixture of positional and geometric isomers of LA in which the double bonds are conjugated. The peak height ratio of CLA to LA was used as a biomarker of lipid peroxidation. GHbA1c, an index of glycemic stress, was measured by high-performance liquid chromatography. There were significantly increased ratios of CLA to LA in diabetic erythrocytes compared with control erythrocytes. These ratios of CLA to LA were also significantly correlated with GHbA1c values. This suggests that glycation via chronic hyperglycemia links lipid peroxidation in the erythrocytes of both diabetic and healthy subjects.

Adult↗
PubMed1998

Solution NMR structure and backbone dynamics of the major cold-shock protein (CspA) from Escherichia coli: evidence for conformational dynamics in the single-stranded RNA-binding site.

The major cold-shock protein (CspA) from Escherichia coli is a single-stranded nucleic acid-binding protein that is produced in response to cold stress. We have previously reported its overall chain fold as determined by NMR spectroscopy [Newkirk, K., Feng, W., Jiang, W., Tejero, R., Emerson, S. D., Inouye, M., and Montelione, G. T. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5114-5118]. Here we describe the complete analysis of 1H, 13C, and 15N resonance assignments for CspA, together with a refined solution NMR structure based on 699 conformational constraints and an analysis of backbone dynamics based on 15N relaxation rate measurements. An extensive set of triple-resonance NMR experiments for obtaining the backbone and side chain resonance assignments were carried out on uniformly 13C- and 15N-enriched CspA. Using a subset of these triple-resonance experiments, the computer program AUTOASSIGN provided automatic analysis of sequence-specific backbone N, Calpha, C', HN, Halpha, and side chain Cbeta resonance assignments. The remaining 1H, 13C, and 15N resonance assignments for CspA were then obtained by manual analysis of additional NMR spectra. Dihedral angle constraints and stereospecific methylene Hbeta resonance assignments were determined using a new conformational grid search program, HYPER, and used together with longer-range constraints as input for three-dimensional structure calculations. The resulting solution NMR structure of CspA is a well-defined five-stranded beta-barrel with surface-exposed aromatic groups that form a single-stranded nucleic acid-binding site. Backbone dynamics of CspA have also been characterized by 15N T1, T2, and heteronuclear 15N-1H NOE measurements and analyzed using the extended Lipari-Szabo formalism. These dynamic measurements indicate a molecular rotational correlation time taum of 4.88 +/- 0.04 ns and provide evidence for fast time scale (taue < 500 ps) dynamics in surface loops and motions on the microsecond to millisecond time scale within the proposed nucleic acid-binding epitope.

Amides↗
PubMed1998

Two-domain reconstitution of a functional protein histidine kinase.

In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C). (223-289); 67 residues] and subdomain B [EnvZ(C).(290-450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an alpha/beta-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

Bacterial Outer Membrane Proteins↗