Geneviève Morrow

Overexpression of the small mitochondrial Hsp22 extends Drosophila life span and increases resistance to oxidative stress.

Heat shock proteins (Hsp) are involved in protein folding, transport and stress resistance. Studies reporting an increased mRNA level of hsp genes in aged Drosophila suggest that expression of Hsp might be beneficial in preventing damages induced by aging. Because oxidative damage is often observed in aged organisms and mitochondria are sensitive to reactive oxygen species, we tested the hypothesis that increased levels of a small Hsp localized in mitochondria, Hsp22 of Drosophila melanogaster, could protect mitochondrial proteins and influence the aging process. We demonstrate that a ubiquitous or a targeted expression of Hsp22 within motorneurons increases the mean life span by more than 30%. Hsp22 shows beneficial effects on early‐aging events since the premortality phase displays the same increase as the mean lifespan. Moreover, flies expressing Hsp22 in their motorneurons maintain their locomotor activity longer as assessed by a negative geotaxis assay. The motorneurons‐targeted expression of Hsp22 also significantly increases flies’ resistance to oxidative injuries induced by paraquat (up to 35%) and thermal stress (39% at 30°C and 23% at 37°C). These observations establish Hsp22 as a key player in cell‐protection mechanisms against oxidative injuries and aging in Drosophila and corroborate the pivotal role of mitochondria in the process of aging.

Expression of Heat Shock Proteins in Mouse Skin During Wound Healing

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Decreased Lifespan in the Absence of Expression of the Mitochondrial Small Heat Shock Protein Hsp22 in Drosophila

Aging is a well regulated biological process involving oxidative stress and macromolecular damages. Three main pathways have been shown to influence lifespan, the insulin/insulin-like growth factor-1 pathway, the silent regulator pathway, and the target of rapamycin pathway. Among many proteins influencing lifespan, two transcription factors, FOXO and the heat shock factor, have been shown to be involved in the aging process and in small heat shock proteins (sHsps) expression following stress and during lifespan. We have recently shown that overexpressing the mitochondrial Hsp22 increases Drosophila melanogaster lifespan by 32% and resistance to oxidative stress. Here we show that flies that are not expressing this mitochondrial small Hsp22 have a 40% decrease in lifespan. These flies die faster than their matched control and display a decrease of 30% in locomotor activity compared with controls. The absence of Hsp22 also sensitizes flies to mild stress. These data support a key role of sHsps in aging and underline the importance of mitochondrial sHsps in this process.

Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Lumbricus terrestris

Abstract Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24–72 hours) and long-term (14–16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.

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°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.

The Small Heat Shock Protein Hsp22 of Drosophila melanogaster Is a Mitochondrial Protein Displaying Oligomeric Organization

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1–17 and an unknown number of residues between 17–28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8–13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 ofDrosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.

Small heat shock protein expression and functions during development

The expression of small heat shock proteins is tightly regulated during development in multiple organisms. As housekeeping proteins, small heat shock proteins help protect cells from apoptosis, stabilize the cytoskeleton and contribute to proteostasis. Consistently, depletion of one small heat shock protein is usually not detrimental due to a certain level of redundancy between the functions of each small heat shock protein. However, while their stress-induced expression is regulated by heat shock factors, their constitutive expression is under the control of other specific transcription factors, suggesting the existence of very specialized functions. This review focuses on the expression patterns and functions of small heat shock proteins in various organisms during development. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Drosophila Small Heat Shock Proteins: Cell and Organelle-Specific Chaperones?

The cellular response to a heat shock treatment was originally observed in Drosophila by the appearance of specific puffs on polytene chromosomes (Ritossa 1962). These puffs are characterised by a high level of transcriptional activity. Concomitant with this physical manifestation is the strong induction of a restricted number of specific polypeptides thereby named Heat Shock Proteins (HSP). In Drosophila melanogaster, the major HSP were first identified through 35S-labelling experiments on Drosophila tissue culture cells and salivary glands (Tissieres et al. 1974) and have been commonly divided into three subfamilies based on their apparent molecular weight on SDS-PAGE. The Hsp83 and Hsp60 species are each sole members of their class, while many different genes encode for the highly conserved members of the Hsp70 subfamily. The small heat shock proteins (sHSP) group includes four polypeptides encoded by identified genes (hsp22, hsp23, hsp26 and hsp27) which are all found within the same locus on chromosome 3 (67B). However, additional genes carrying open reading frames (ORF) which could potentially encode for proteins carrying the α-crystallin domain, hallmark domain of the sHSP family, have been readily identified both within (hsp67a, hsp67b and hsp67c—formerly known as gene1, gene2 and gene3) and outside (l(2)efl) the 67B locus.

Effects of different small HSPB members on contractile dysfunction and structural changes in a Drosophila melanogaster model for Atrial Fibrillation

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pre-treatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition, tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling.

Proproliferative Functions of Drosophila Small Mitochondrial Heat Shock Protein 22 in Human Cells

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (>30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598–599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382–43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated β-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.