Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.
RraA and RraB are recently discovered protein inhibitors of RNAse E, which forms a large protein complex termed the degradosome that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. Here, we report that these E. coli protein inhibitors physically interact with RNAse ES, a Streptomyces coelicolor functional ortholog of RNAse E, and inhibit its action in vivo as well as in vitro; however, unlike their ability to differentially modulate E. coli RNAse E action in a substrate-dependent manner by altering the composition of the degradosome, both proteins appear to have a general inhibitory effect on the ribonucleolytic activity of RNAse ES, which does not interact with E. coli polynucleotide phosphorylase, a major component of the degradosome. Our findings suggest that these regulators of RNAse activity have a conserved intrinsic property enabling them to directly act on RNAse E-related enzymes and inhibit their general ribonucleolytic activity.
Escherichia coli RNase e contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase e, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. studies of cleavage of fluorogenic substrate and eMsA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65 hGFLpLK 71 -which includes amino acids previously implicated in substrate binding and catalysisand 24 LYDLDIespGheQK 37 -which includes the Q36 locus-were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase e locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding
e 2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant ⌬65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the ⌬193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5=-dithio-bis-(2-nitrobenzoic acid) and ZnCl 2 , which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higherorder oligomers at high pH, high temperature, or in the presence of H 2 O 2 . SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.
RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNA Asn precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity-implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.
RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5′ terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.
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