Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
...
PMID:HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. 865 Jan 74

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.
...
PMID:Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli. 866 28

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.
...
PMID:Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. 874 35

Hs1VU in E. coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain beta-type subunits of the 20S proteasome. Here we show that the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome. However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself. Instead, it stimulated the hydrolysis of ATP by HslU several-fold. Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine. Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV. These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU.
...
PMID:Poly-L-lysine activates both peptide and ATP hydrolysis by the ATP-dependent HslVU protease in Escherichia coli. 895 32

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.
...
PMID:The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. 903 94

Recent studies of the 20S proteasome from Thermoplasma acidophilum have uncovered some fundamental new properties of its catalytic mechanism. Unlike conventional proteases, 20S and 26S proteasomes degrade protein substrates in a highly processive fashion. They cleave a protein substrate to small peptides before attacking another substrate molecule. This processive behavior is an inherent feature of the 20S particle not requiring cofactors or ATP hydrolysis. Recently, we have described a proteasome-like particle, HslVU, in Escherichia coli. HslVU is a two-component ATP-dependent protease composed of the proteasome-related peptidase HslV (beta-subunit) and the ATPase HslU. In active HslVU complex, cleavage of small peptides and proteins requires the presence of ATP. EM analysis revealed that HslV and HslU are both ring-shaped particles and that the active HslVU complex is a cylindrical four-ring structure, composed of HslV, a two-ring dodecamer, sandwiched between HslU rings. Elucidation of its mode of action may help us understand the role of ATP in function of the 26S proteasome. Several proteasome-specific inhibitors have been recently identified which block the function of proteasome in vivo. These agents have proven very useful to clarify the intracellular function of the proteasome. In mammalian cells, both the rapid degradation of short-lived regulatory proteins and of abnormal polypeptides and the slower degradation of long-lived proteins are blocked by these agents. Thus, in mammalian cells, the proteasome is the site for the degradation of most cell proteins. In contrast, in budding yeast, proteasome inhibitors block the degradation of short-lived proteins but not the breakdown of long-lived proteins, which can be blocked by inhibitors of vacuolar proteases. The inhibition of proteasome function in yeast and mammalian cells, presumably by causing an accumulation of unfolded proteins, triggers the expression of heat shock proteins and concomitantly increases cell resistance to high temperature and various toxic insults.
...
PMID:New insights into the mechanisms and importance of the proteasome in intracellular protein degradation. 916 63

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.
...
PMID:Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle. 922 88

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.
...
PMID:Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease. 925 89

HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase. Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins. We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g. casein). Also, the nonhydrolyzable analogs of ATP, 5'-adenylyl beta, gamma-imidodiphosphate (AMP-PNP) and adenosine 5'-(alpha, beta-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP. This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together. Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog. The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP. NH4+ and Cs+ had similar effects as K+, but Na+ and Li+ were ineffective. AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K+, Cs+, or NH4+, AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed. In addition, MgCl2, MnCl2, and CaCl2 allowed some peptidase and caseinase activity in the absence of any nucleotide. However, Mn2+ and Ca2+, unlike Mg2+, abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP. These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process. Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites. However, AMP-PNP binding together with K+ can induce a form of HslVU that degrades proteins without energy consumption.
...
PMID:Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP hydrolysis. 926 Nov 50

The PA700-like proteasome activator complex was highly purified from porcine erythrocytes, and its properties were compared with those of the regulatory complex disassembled from the purified 26S proteasome. The molecular mass of the PA700-like complex, which comprises 25-110-kDa subunits, was estimated to be 800 kDa by Superose 6 gel filtration. This complex showed neither ATPase activity nor peptidase activity toward Suc-Leu-Leu-Val-Tyr-MCA. Nevertheless, it was possible to make a high molecular mass complex from the purified PA700-like complex by incubating with the 20S proteasome in the presence of ATP. In contrast, the regulatory complex dissociated from the 26S proteasome did not reconstitute a larger complex under the same conditions. The subunit composition of the PA700-like complex was similar but not identical to that of the regulator complex dissociated from the 26S proteasome: the former complex had a 25-kDa subunit which is absent in the latter, whereas the latter had two or three 43-kDa subunits lacking in the former. These results indicate that the purified PA700-like proteasome activator complex is structurally and functionally distinct from the regulatory complex dissociated from the 26S proteasome, implying the involvement of modulating factors in the 26S proteasome assembly.
...
PMID:Difference between PA700-like proteasome activator complex and the regulatory complex dissociated from the 26S proteasome implies the involvement of modulating factors in the 26S proteasome assembly. 927 59


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---