Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase) can be described by three slow kinetic phases. The slowest phase is absent in the P117G mutant of SNase. Peptidyl prolyl cis-trans isomerase (cyclophilin), which has been shown to catalyze the slow folding reactions of some proteins, was employed to determine which of the refolding reactions of SNase and P117G SNase involve proline isomerization. We report here that all three folding phases of the wild type and the slower phase of P117G SNase are catalyzed by prolyl isomerase, indicating that proline isomerization is involved in these fluorescence-detected phases in the refolding of SNase. Since the rates of these phases are denaturant-dependent, we conclude that the slow folding steps involve isomerization of non-native cis proline peptide bonds and are tightly coupled to denaturant-sensitive structural changes.
 ...
PMID:Effect of prolyl isomerase on the folding reactions of staphylococcal nuclease. 939 41

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.
 ...
PMID:Effects of proline mutations on the folding of staphylococcal nuclease. 1002 6

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.
 ...
PMID:Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases. 1646 29

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.
 ...
PMID:An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease. 2779 19