Studies of the small heat shock proteins of <i>Caenorhabditis elegans</i> using anti‐peptide antibodies

Hockertz, Michael K.; Clark‐Lewis, Ian; Candido, E. P. M.

doi:10.1016/0014-5793(91)80335-z

Cited by 26 publications

(12 citation statements)

References 17 publications

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“…2A). The estimated size of the complex is essentially identical to that of HSP16-2 isolated from a heat-shocked nematode extract (71). Interestingly, the H 6 HSP16-2 protein also assembles into large oligomeric complexes of somewhat larger size (680 kDa), as judged by SEC (Fig.…”

Section: Hsp16-2 Quaternary Structure and Subunit Orientationsupporting

confidence: 49%

Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

Leroux¹,

Melki²,

Gordon³

et al. 1997

Journal of Biological Chemistry

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206

197

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The small heat shock protein (smHSP) and ␣-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the ␣-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the Nterminal domain but not most of the the C-terminal extension which follows the ␣-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partially or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated across a membrane, or damaged by conditions of cellular stress. Many chaperones are expressed at higher levels during biological stresses, and are members of heat shock protein (HSP) 1 families (1-4). Whereas some chaperones (HSP70, HSP40, and HSP60) are involved in protein folding under normal conditions in vivo (5-7), others such as HSP104 (8 -10), inducible HSP70s (11), and small HSPs (12-15) are known to play important roles in protecting organisms from stress.The small HSPs (smHSPs) form a structurally divergent protein family with members present in Archaea, Bacteria, and Eukarya (16, 17). The presence of an evolutionarily conserved ␣-crystallin domain distinguishes all smHSPs and ␣-crystallins (18 -20). This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension. Some smHSP genes contain an intron which delineates the N-terminal and ␣-crystallin domains (21, 22), and structural studies support a two-domain structure (20,23,24) consisting mostly of ␤-sheets (25, 26) for smHSPs. The C-terminal extensions of smHSPs appear relatively unstructured (27,28) and are known to undergo numerous modifications, including truncati...

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Section: Hsp16-2 Quaternary Structure and Subunit Orientationsupporting

confidence: 49%

Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

Leroux¹,

Melki²,

Gordon³

et al. 1997

Journal of Biological Chemistry

Self Cite

206

197

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“…5B, lanes 3 and 4). In contrast, identical cross-linking experiments performed with HSP16 -2, which forms high M r complexes (48), resulted in efficient cross-linking of multimers at low smHSP concentrations in the presence of competitor BSA. 3 To determine whether the elution behavior of HSP12.6 from a size exclusion column is due to dimerization or to an extended, nonglobular conformation, we carried out sedimenta-3 M. R. Leroux, R. Melki, B. Gordon, G. Batelier, and E. P. M. Candido, submitted for publication.…”

Section: Resultsmentioning

confidence: 90%

Unique Structural Features of a Novel Class of Small Heat Shock Proteins

Leroux

Brian

Batelier

et al. 1997

Journal of Biological Chemistry

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101

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Small heat shock proteins (smHSPs) and ␣-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa. Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes. Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans. Like other members of its class, HSP12.6 possesses a conserved ␣-crystallin domain but has the shortest N-and C-terminal regions of any known smHSP. Expression of HSP12.6 is limited to the first larval stage of C. elegans and is not significantly upregulated by a wide range of stressors. Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo. HSP12.6 was produced in Escherichia coli as a soluble protein and purified. Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination. Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate. The structural and functional implications of these findings are discussed.

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“…Images A6 and A7 are merged in A8. (B) HSP16 localization with polyclonal anti-HSP16-2 antibody [15]. A9 and A10, staining pattern of HSP12s in vulval region.…”

Section: Resultsmentioning

confidence: 99%

Association of several small heat-shock proteins with reproductive tissues in the nematode Caenorhabditis elegans

Ding

Candido

2000

Biochemical Journal

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Studies of the small heat shock proteins of Caenorhabditis elegans using anti‐peptide antibodies

Cited by 26 publications

References 17 publications

Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

Unique Structural Features of a Novel Class of Small Heat Shock Proteins

Association of several small heat-shock proteins with reproductive tissues in the nematode Caenorhabditis elegans

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