Pneumococcal HtrA protease mediates inhibition of competence by the CiaRH two-component signaling system.

Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.

Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex.

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site for target proteases; however, the complete role of the reactive loop in determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the extent to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha-1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing length taken from the P6-P3' region of PI replaced the corresponding residues of ACT. The effectiveness of each chimera as an inhibitor of HNE was assessed by measuring (1) the rate of inhibitory complex formation and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-like proteases, the series of chimeras showed no consistent progress toward the production of an inhibitor with the inhibitory properties of PI. The most rapid complex formation and most stable complexes were observed for chimeras with the P3-P1 residues of PI, whereas extending the replacement region to the P6 residue resulted in a considerable decrease in both inhibitory parameters. In order to study two additional features of the PI reactive loop that may play a role in the presentation of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an inhibition rate constant at least 60-fold less than that observed for PI inhibition of HNE and the most long lived chimera-HNE complexes broke down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one serpin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in determining serpin inhibitory activity against target proteases.

Diverse effects of pH on the inhibition of human chymase by serpins.

Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.

Arginine substitutions in the hinge region of antichymotrypsin affect serpin beta-sheet rearrangement.

A hallmark of serpin function is the massive beta-sheet rearrangement involving the insertion of the cleaved reactive loop into beta-sheet A as strand s4A. This structural transition is required for inhibitory activity. Small hydrophobic residues at P14 and P12 positions of the reactive loop facilitate this transition, since these residues must pack in the hydrophobic core of the cleaved serpin. Despite the radical substitution of arginine at the P12 position, the crystal structure of cleaved A347R antichymotrypsin reveals full strand s4A insertion with normal beta-sheet A geometry; the R347 side chain is buried in the hydrophobic protein core. In contrast, the structure of cleaved P14 T345R antichymotrypsin reveals substantial yet incomplete strand s4A insertion, without burial of the R345 side chain.

Distortion of the active site of chymotrypsin complexed with a serpin.

There is no complete understanding of how serine protease inhibitors of the serpin family inhibit their target enzymes. Structural and biochemical studies have suggested that serpins utilize a mechanism that is distinct from the standard mechanism of inhibition proposed for most small protein protease inhibitors. Proton nuclear magnetic resonance spectroscopy was used in the present study to demonstrate a fundamental difference in the atomic environment of the catalytic triad of enzyme in complex with serpins when compared to uncomplexed enzyme and enzyme in complex with standard mechanism inhibitors. This work demonstrates that the active site of chymotrypsin is distorted when complexed to a serpin and makes tenable a mechanism of inhibition in which the serpin induces a conformational change in the enzyme that dramatically reduces or completely abrogates the catalytic activity of the protease.

Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin.

The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretary granules, and the interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase-SL interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme-inhibitor complex. The dissociation equilibrium constant (determined in reactions containing 0.18 M or 1.0M NaCl (pH 8.0, 25 degrees C) was 5 X 10(-8) and 2 x 10(-8) M, respectively. Addition of heparin to the low-salt reaction decreased the Ki approximately 10-fold to a value of 3 x 10(-9) M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10-fold increase in the association rate constant (kass) from 2 X 10(4) to 3 X 10(5) M-1 s-1. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitor in vivo, especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI.

Conversion of alpha 1-antichymotrypsin into a human neutrophil elastase inhibitor: demonstration of variants with different association rate constants, stoichiometries of inhibition, and complex stabilities.

Despite the homology with alpha 1-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibitor of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutrophil elastase (HNE) with wild-type recombinant ACT and recombinant variants of ACT were studied. ACT variants were generated where (1) the primary interaction site, the P1 position, was replaced with the P1 residue of alpha 1PI, (2) the residues corresponding to P3-P3' were replaced with those of alpha 1PI, and (3) the residues corresponding to the canonical recognition sequence as well as flanking residues encompassing the exposed reactive loop of the inhibitor were replaced with the corresponding residues of alpha 1PI. Each variant was analyzed to determine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate constant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiometry of inhibition, SI), and (3) the stability of the enzyme-serpin complex. Replacing Leu with Met in the P1 position (rACT-L358M) was sufficient to convert rACT into an inhibitor of HNE with an apparent second-order rate constant (k'/[I]) of 4 x 10(4) M-1 s-1 and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in the reactive loop.(ABSTRACT TRUNCATED AT 250 WORDS)

cis-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone.

In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site.