Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase.

Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.

Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP.

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.

Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP.

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

Assembly of both the head and tail of bacteriophage Mu is blocked in Escherichia coli groEL and groES mutants.

Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504-5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.

Bacteriophage Mu head assembly.

The protein composition of defective particles produced by various bacteriophage Mu head-gene mutants was analyzed by SDS-PAGE. An abundant 20-kDa protein was detected in only one type of defective head. This protein exhibits properties of a scaffolding protein. A 50-kDa structural protein present in most defective heads was shown to be produced by cleavage of the C-terminus of the 64-kDa polypeptide encoded by gene H. Cleavage occurs during head assembly at a site which, according to earlier results, might separate two different functional domains in gpH. A fraction of the gpH molecules produced upon Mu induction sediment in a 25 S complex, suggesting that gpH participates in the formation of an early intermediate of Mu head assembly. Characteristics of gpH suggest that it may be the Mu portal protein.

Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease.

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.

Dog gastric lipase: stimulation of its secretion in vivo and cytolocalization in mucous pit cells.

Dog gastric lipase (DGL) secretion is stimulated in vivo by urecholine, pentagastrin, histamine, 16,16-dimethyl prostaglandin E2, and secretin. Under fasting conditions, DGL is irreversibly inactivated by gastric acid below pH 1.5; consequently, DGL output can be underestimated. This problem has been resolved by buffering the acid or by using an antisecretory drug such as omeprazole during stimulation. There is a clear parallelism between the secretion of DGL and of gastric mucus. This observation led to the present investigation of the cellular localization of DGL using immunofluorescence techniques. Results showed that DGL is cytolocalized in mucous pit cells of gastric glands. Pepsinogen is found in chief cells. To the authors' knowledge, this is the first description of an enzyme (gastric lipase) secreted by mucous-type gastric cells. In contrast to other species, gastric lipase of the dog is located in cardiac, fundic, and antral mucosae.

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.