Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli

Chuang, Shuang‐En; Burland, Valerie; Plunkett, Guy; Daniels, Donna L.; Blattner, Frederick R.

doi:10.1016/0378-1119(93)90167-2

Cited by 157 publications

(132 citation statements)

References 11 publications

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“…2A). Because both ibpA and ibpB are located in one operon in E. coli (37) and it was shown that sHsps from other bacteria form heterooligomers (34), it is plausible that IbpA and IbpB cooperate in substrate stabilization during thermal stress. Therefore, we purified IbpB (Fig.…”

Section: Resultsmentioning

confidence: 99%

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The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

Matuszewska

Kuczyńska-Wiśnik

Laskowska

et al. 2005

Journal of Biological Chemistry

101

104

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The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate.The proper conformation of proteins is challenged by stress conditions. Exposure to extreme heat shock conditions results in a massive aggregation of proteins inside both prokaryotic and eukaryotic cells (1-3). Chaperones from the Hsp100 family, that is ClpB in Escherichia coli and Hsp104 in the yeast Saccharomyces cerevisiae, were implicated in the disaggregation reaction, because aggregated proteins were not eliminated in either clpB or HSP104 deletion strains (1-3). Additionally, the clpB and HSP104 gene products were identified as factors conferring thermotolerance in E. coli and S. cerevisiae (4 -7). However, in vitro studies on the reactivation of aggregated proteins showed that chaperones from the Hsp100 family alone are not sufficient for disaggregation and refolding. Other chaperone proteins are also involved in this process. E. coli Hsp70 (DnaK) and its cochaperones (DnaJ and GrpE) cooperate with ClpB and form a bi-chaperone system capable of efficient disaggregation of aggregated proteins (3,8,9). Analogous Hsp100-Hsp70 bi-chaperone systems able to disaggregate denatured protein substrates in vitro were established using chaperones from other bacterial species (10), as well as from yeast cytosol (11) and mitochondria (12, 13). However, the efficiency of refolding reaction catalyzed by these bichaperone systems depends strongly on the physical properties of protein aggregates. It was proposed that small heat shock proteins (sHsps) 1 associate with aggregated proteins and change their physical properties in such a way that chaperone-mediated disaggregation and refolding become much more efficient (14 -19). However, little is known about the molecular mechanism of these processes.Small heat shock proteins are widely distributed both in prokaryotes and eukaryotes. Members of this diverse protein family are characterized by relatively low monomeric molecular masses (15-43 kDa) and a conserved stretch of ϳ100 amino acid residues (reviewed in Refs. 20 and ...

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Section: Resultsmentioning

confidence: 99%

“…Two members of the sHsp family, IbpA and IbpB, are present in E. coli. The IbpA and IbpB proteins are 48% identical at the amino acid sequence level (37), and both were identified as proteins associated with inclusion bodies (35). IbpA/IbpB were also found in aggregated protein fractions following heat stress (2).…”

mentioning

confidence: 99%

The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

Matuszewska

Kuczyńska-Wiśnik

Laskowska

et al. 2005

Journal of Biological Chemistry

101

104

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“…The HslV gene product is a peptidase that shows homology to ␤ type subunits of the proteasome, including the presence of a NH 2 -terminal threonine (15,14). HslV is cotranscribed with the adjacent HslU gene that encodes an ATPase with homology to other known Escherichia coli ATPases such as ClpX (50% identity) (16). Together the HslV and HslU gene products make up a complex with an ATP-dependent proteolytic activity similar to that of the eukaryotic proteasome (14).…”

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confidence: 99%

Covalent modification of the active site threonine of proteasomal β subunits and theEscherichia colihomolog HslV by a new class of inhibitors

Bogyo

McMaster

Gaczyńska

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

430

336

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The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L 3 VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L 3 VS and a 125 I-labeled nitrophenol derivative ( 125 I-NIP-L 3 VS) covalently modify the active site threonine of the catalytic ␤ subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the ␤ subunit's NH 2 terminus. 125 I-NIP-L 3 VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV͞HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.

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“…HslVU is of particular interest as a simple model system for the eukaryotic 26S proteasome (3)(4)(5). Subunits of HslV share significant sequence (6) and structural similarity (7) with the proteasomal ␤-subunits but form different oligomeric assemblies with D6 symmetry of HslV and D7 symmetry of the archaeal ␤-subcomplex. HslU, a member of the Hsp100 family of ATPases (8), exhibits both ATPase (3) and chaperone activities (9).…”

mentioning

confidence: 99%

Mutational studies on HslU and its docking mode with HslV

Song

Hartmann

Ramachandran

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

133

124

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HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition͞translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the ''sensor arginine'' (R393) and the ''arginine finger'' (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

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Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli

Cited by 157 publications

References 11 publications

The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

Covalent modification of the active site threonine of proteasomal β subunits and theEscherichia colihomolog HslV by a new class of inhibitors

Mutational studies on HslU and its docking mode with HslV

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