Dataset Information

Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster.

ABSTRACT: The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42 degrees C, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.

SUBMITTER: Morrow G

PROVIDER: S-EPMC1400613 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster.

Morrow Geneviève G Heikkila John J JJ Tanguay Robert M RM

Cell stress & chaperones 20060101 1

The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in t ...[more]

PMID: 16572729

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Project description:It is known from earlier studies that the heat shock (HS) response in Malpighian tubules (MTs) of Drosophila larvae is different from that in other tissues because instead of the Hsp70 and other common heat shock proteins, Hsp64 and certain other new proteins are induced immediately after HS. In the present study, we examined the kinetics of the synthesis of Hsp70 and Hsp64 immediately after HS and during recovery from HS by 35S-methionine labeling and Western blotting. In addition, we also examined the transcriptional activity of hsp70 genes in larval MT cells at different times after HS by in situ hybridization and Northern blotting. The HS-induced synthesis of Hsp64 ceased by 1 hour of recovery from the HS when synthesis of the Hsp70 commenced. Our results revealed that the induced synthesis of Hsp64 immediately after HS was dependent on new transcription. Although the levels of Hsp70 in MT cells rapidly increased after its synthesis began during recovery, the levels of Hsp64 remained unaltered irrespective of its new synthesis occurring during or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the hsp70 genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3' untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the beta-galactosidase activity in 2 transgenic lines carrying the LacZ reporter gene under hsp70 promoter and different lengths of the 5'UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5'UTR.

Project description:Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

Dataset Information

Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster.

Publications

Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster.

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