The OmpR-family of proteins: insight into the tertiary structure and functions of two-component regulator proteins

The Escherichia coli DNA-binding protein OmpR is the best characterized of those regulator proteins making up "two-component system," the simplest known form of bacterial signal transduction systems. Previous inspections of the E. coli genome DNA sequences have revealed that there are 15 proteins whose amino acid sequences show extensive similarities to that of OmpR (the OmpR-family of proteins). The three-dimensional structures of several OmpR-family proteins have been determined. In this review, we investigated the structures and amino acid sequences of this family of proteins. The results reveal several notable conservative varieties in their tertiary structures and functions.     

A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves

We purified a glutamyl endopeptidase that is a major foliar endopeptidase in cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degrees C, respectively. An internal amino acid sequence of the endopeptidase was highly homologous to a partial sequence of unidentified proteins deduced from genetic information for Arabidopsis thaliana, soybean and rice, but not to the sequences of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distributed only among plant species. The activity of the cucumber glutamyl endopeptidase was inhibited by at least three inhibitors existing in cucumber leaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which did not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

A full biological response to autoantibodies in Graves' disease requires a disulfide-bonded loop in the thyrotropin receptor N terminus homologous to a laminin epidermal growth factor-like domain

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.     

Complementing yeast rho1 mutation groups with distinct functional defects

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.     

Association between a single-nucleotide polymorphism in the promoter of the human interleukin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two genetic loci have on susceptibility to the disease

Genetic variants of interleukin-3 (IL-3), a well-studied cytokine, may have a role in the pathophysiology of rheumatoid arthritis (RA); but reports on this association sometimes conflict. A case-control study was designed to investigate association between RA and a single-nucleotide polymorphism (SNP) in the IL-3 promoter region. Comparison of cases of RA versus control individuals yielded a chi(2) value of 14.28 (P=.0002), with a genotype odds ratio of 2.24 (95% confidence interval [95%CI] 1.44-3.49). When female cases with earlier onset were compared with female control individuals, the SNP revealed an even more significant correlation, with chi2=21.75 (P=.000004) and a genotype odds ratio of 7.27 (95%CI 2.80-18.89). The stronger association that we observed in this clinically distinct subgroup (females with early onset), within a region where linkage disequilibrium was not significantly extended, suggested that the genuine RA locus should locate either within or close to the IL-3 gene. Combined genotype data on SNPs on eight other candidate genes were combined with our IL-3 results, to estimate relationships between pairs of loci and RA, by maximum-likelihood analysis. The utility of combining the genotype data in this way to identify possible contributions of various genes to this disease is discussed.     

Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.     

A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity

A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate. The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins. The median effective concentration of this protein was 0.23 microg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins. The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.     

Thermostabilization of a chimeric enzyme by residue substitutions: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase

A chimeric 3-isopropylmalate dehydrogenase, named 2T2M6T, made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, was found to be considerably more labile than the T. thermophilus wild-type isopropylmalate dehydrogenase. In order to identify the molecular basis of the thermal stability of the T. thermophilus isopropylmalate dehydrogenase, 11 amino acid residues in the mesophilic portion of the chimera were substituted by the corresponding residues of the T. thermophilus enzyme, and the effects of the side chain substitutions were analyzed by comparing the reaction rate of irreversible heat denaturation and catalytic parameters of the mutant chimeras with those of the original chimera, 2T2M6T. Four single-site mutants were successfully stabilized without any loss of the catalytic function. All these four sites are located in loop regions of the enzyme. Our results strongly suggest the importance of these loop structures to the extreme stability of the T. thermophilus isopropylmalate dehydrogenase.