Mitochondrial Heat Shock Protein Research Articles

Differential Effects of Mitochondrial Heat Shock Protein 60 and Related Molecular Chaperones to Prevent Intracellular β-Amyloid-induced Inhibition of Complex IV and Limit Apoptosis

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor‐associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.

Mitochondrial Heat Shock Protein 60 Is Increased in Cerebrospinal Fluid following Pediatric Traumatic Brain Injury

Mitochondrial dysfunction occurs after traumatic brain injury (TBI) and contributes significantly to subsequent cell death. Heat shock protein 60 (hsp60) is a predominantly mitochondrial protein with important homeostatic functions. Induction of hsp60 has been demonstrated in cerebral ischemia models, possibly reflecting mitochondrial stress. We measured hsp60 concentration in the cerebrospinal fluid (CSF) of 34 infants and children after severe TBI and of 7 control patients by ELISA. Peak CSF hsp60 concentration was increased in TBI patients versus controls (0.84 ng/ml, range 0–44.59, vs. 0.0 ng/ml, range 0–0.48; p < 0.05). Induction of hsp60 occurred early after the injury. Peak hsp60 concentration was independently associated with the severity of injury, defined as the admission Glasgow Coma Scale score. These data suggest that increased hsp60 in CSF might reflect the severity of early mitochondrial stress or damage after TBI.

Region-specific induction of hypoxic tolerance by expression of stress proteins and antioxidant enzymes

We examined the induction of hypoxic tolerance after hypoxic preconditioning in the frontal cortex, caudate putamen and thalamus using the dynamic positron autoradiography technique and [18F]2-fluoro-2-deoxy-D-glucose with rat brain slices. Hypoxic tolerance induction was confirmed in the frontal cortex, but not in the caudate putamen and thalamus. Next, we compared the gene expression in the frontal cortex and caudate putamen using the ATLAS Rat Stress Array, and found that the expression of 150-kDa oxygen-regulated protein and mitochondrial heat shock protein 70 as stress proteins, and copper-zinc-containing superoxide dismutase and manganese-containing superoxide dismutase as antioxidant enzymes was elevated only in the frontal cortex. These results suggest that the induction of hypoxic tolerance after hypoxic preconditioning is region-specific, and stress proteins and antioxidant enzymes participate in this phenomenon.

Iron chelator desferrioxamine induces decrease of mitochondrial heat shock protein TNF receptor‐associated protein 1(TRAP1) in Chang cells

Heat shock proteins (HSPs) are molecular chaperones which regulate diverse cellular functions e.g. apoptosis and cell cycle. Tumour necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial HSP and has been reported that it may play roles in apoptosis as well as differentiation. But the exact roles of TRAP1 remain unclear. It has been implicated that iron chelator desferroxamine (DFO) induces mitochondrial dysfunction such as decrease of oxidative phosphorylation as well as change of cellular morphology. We used DFO to examine the mitochondrial morphology, function, and levels of related mitochondrial HSPs. Treatment of DFO increased transferrin mRNA but not aconitase1 and transferrin receptor1 in human hepatocyte cell line Chang cells. In addition, DFO induced G0/G1 cell cycle arrest, abnormal morphology of mitochondria, decrease of NADH-dependent dehydrogenase activity, and disruption of mitochondrial membrane potential (ΔΨm). Also we found decrease of TRAP1 at the mRNA and protein level in DFO-treated Chang cells, but not other mitochondrial HSPs such as HSP60, mtHSP70. This result suggests that TRAP1 might have an important role to maintain mitochondrial integrity.

Mitochondrial Protection From Ischemia‐Reoxygenation Injury with Mitochondrial Heat Shock Protein 70 Overexpression

The majority of mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence. These preproteins traverse the outer mitochondrial membrane in an unfolded state, and are then translocated through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mtHsp70). In this study, neonatal rat cardiac myocytes (NCM) were infected with an adenoviral vector expressing mtHsp70 or an empty control (Adv-) for 48 hours, and submitted to 8 hours of simulated ischemia (hypoxia) followed by 16 hours of reperfusion (reoxygenation). Infection with mtHsp70 virus yielded a large increase in mtHsp70 protein in NCM mitochondria as compared to Adv-. Cell viability following simulated I/R was greater in mtHsp70 myocytes (P<0.05). Simulated I/R caused a significant increase in reactive oxygen species (ROS) generation in Adv-infected myocytes (P<0.05), which was not observed in mtHsp70-infected myocytes. Mitochondrial complex III and IV activities were greater in mtHsp70-infected myocytes following simulated I/R as compared to Adv- (P<0.05 and P<0.01, respectively). Following simulated I/R, ATP content was greater in mtHsp70-infected cells, as compared to Adv- (P<0.05). Further, caspase-3 activity was lower in mtHsp70-infected myocytes as compared to Adv- after simulated I/R (P<0.05). These results indicate that overexpression of mtHsp70 provides protection to the mitochondria against damage resulting from simulated I/R injury, which may be due in part to a decrease in ROS generation, preserving mitochondrial complex function and ATP formation.

Therapeutic Approach for Diabetic Nephropathy Using Gene Delivery of Translocase of Inner Mitochondrial Membrane 44 by Reducing Mitochondrial Superoxide Production

Hyperglycemia-induced overproduction of mitochondrial reactive oxygen species has emerged as a major player in diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane 44 (TIM44) was identified by upregulation in diabetic mouse kidneys. TIM44 functions as a membrane anchor of mitochondrial heat-shock protein 70 (mtHsp70) to TIM23 complex and is involved in the import of mitochondria-targeted preproteins into mitochondrial matrix. The process is dependent on inner membrane potential and ATP hydrolysis on ATPase domain of mitochondrial heat-shock protein 70. Hemagglutination virus of Japan-envelope vector that carries pcDNA3.1 plasmid that contains the full-length cDNA of TIM44 and control plasmid were injected weekly into the tail vein of uninephrectomized streptozotocin-induced diabetic CD-1 mice. The gene delivery alleviated proteinuria and renal hypertrophy at 8 wk after the injection, inhibited renal cell proliferation and apoptosis, and suppressed superoxide production. In vitro experiments, using human proximal tubular (HK2) cells, revealed that the gene delivery of TIM44 reversed high glucose-induced metabolic and cellular abnormalities such as enhanced reactive oxygen species production, increased ATP contents, alterations in inner membrane potential, increased cell proliferation, and apoptosis. Transfection with siRNA and expressing vector of TIM44 revealed that TIM44 facilitates import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase into mitochondria. The gene delivery of TIM44 therefore seems to be beneficial for the maintenance of mitochondrial function and is a novel therapeutic approach for diabetic nephropathy.

On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60

The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular "brotherhood", their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.

Death-signal-induced relocalization of cyclin-dependent kinase 11 to mitochondria

Fas receptor-Fas ligand interaction appears to be important in carcinogenesis, tumour outgrowth and metastasis. Emerging evidence suggests that CDK11 (cyclin-dependent kinase 11) plays a role in apoptosis and melanoma development. Here, we show that CDK11p110 protein kinase was cleaved after induction of apoptosis by Fas. The N-terminal portion of CDK11p110, CDK11p60, was translocated from the nucleus to the mitochondria. The targeting of CDK11p60 to mitochondria occurred as early as 12 h after treatment. Overexpression of EGFP (enhanced green fluorescent protein)-tagged CDK11p60 could partially break down the mitochondrial membrane potential, induce cytochrome c release and promote apoptosis. Reduction of endogenous CDK11p110 protein levels with siRNA (small interfering RNA) resulted in the suppression of both cytochrome c release and apoptosis. In addition, subcellular fractionation studies of Fas-mediated apoptosis demonstrated that CDK11p60 was associated with the mitochondrial import motor, mitochondrial heat shock protein 70. Taken together, our data suggest that CDK11p60 can contribute to apoptosis by direct signalling at the mitochondria, thereby amplifying Fas-induced apoptosis in melanoma cells.

Physiological and Molecular Effects of 24-Epibrassinolide, a Brassinosteroid on Thermotolerance of Tomato

Brassinosteroids are naturally occurring plant growth regulators, which exhibit structural similarities to animal steroid hormones. Recent studies have indicated that besides an essential role in plant growth and development, brassinosteroids also exert anti-stress effects on plants. We show here that tomato plants treated with 24-epibrassinolide (EBR) are more tolerant to high temperature than untreated plants. An analysis of mitochondrial small heat shock proteins (MT-sHSP) in tomato leaves by western blotting revealed that the MT-sHSP did not preferentially accumulate in EBR treated plants at 25 °C. However, treatment of plants at 38 °C induced much more accumulation of MT-sHSP in EBR treated than in untreated plants. Results of this study provide the first direct evidence for EBR induced expression of MT-sHSP, which possibly induced thermotolerance in tomato plants. EBR treated tomato plants had better photosynthetic efficiency. We also observed significantly higher in vitro pollen germination, enhanced pollen tube growth and low pollen bursting in the presence of EBR at 35 °C, a temperature high enough to induce heat-stress symptoms in tomato, indicating a possible role of EBR during plant reproduction.

Antimelanoma Activity of Apoptogenic Carbonyl Scavengers

Therapeutic induction of apoptosis is an important goal of anticancer drug design. Cellular carbonyl stress mediated by endogenous reactive carbonyl species (RCS) such as glyoxal and methylglyoxal (MG) affects proliferative signaling and metastasis of human tumor cells. Recent research suggests that RCS produced constitutively during increased tumor cell glycolysis may be antiapoptotic survival factors and thus represent a novel molecular target for anticancer intervention. Here, we demonstrate the tumor cell-specific apoptogenicity of carbonyl scavengers, which act by covalently trapping RCS, against human (A375, G361, and LOX) and murine (B16) melanoma cell lines. A structure-activity relationship study identified nucleophilic carbonyl scavenger pharmacophores as the functional determinants of apoptogenic antimelanoma activity of structurally diverse agents such as 3,3-dimethyl-D-cysteine and aminoguanidine. Previous work has demonstrated that covalent adduction of protein-arginine residues in the mitochondrial permeability transition (MPT) pore and heat shock protein 27 by intracellular MG produced in tumor cell glycolysis inhibits mitochondrial apoptosis and enhances cancer cell survival. Indeed, in various melanoma cell lines, carbonyl scavenger-induced apoptosis was antagonized by pretreatment with the membrane-permeable RCS phenylglyoxal (PG). Carbonyl scavenger-induced apoptosis was associated with early loss of mitochondrial transmembrane potential, and cyclosporin A antagonized the effects of carbonyl scavengers, suggesting a causative role of MPT pore opening in carbonyl scavenger apoptogenicity. Consistent with RCS inhibition of mitochondrial apoptosis in melanoma cells, staurosporine-induced apoptosis also was suppressed by PG pretreatment. Our results suggest that carbonyl scavengers acting as direct molecular antagonists of RCS are promising apoptogenic prototype agents for antimelanoma drug design.

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.

Inactivation of the Mitochondrial Heat Shock Protein Zim17 Leads to Aggregation of Matrix Hsp70s Followed by Pleiotropic Effects on Morphology and Protein Biogenesis

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.

Role of cytochrome P-450 in schisandrin B-induced antioxidant and heat shock responses in mouse liver

In order to explore the role of cytochrome P-450 ( P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 ( P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.

Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle

Severe high-altitude hypoxia exposure is considered a triggering stimulus for redox disturbances at distinct levels of cellular organization. The effect of an in vivo acute and severe hypobaric hypoxic insult (48 h at a pressure equivalent to 8,500 m) on oxidative damage and respiratory function was analyzed in skeletal muscle mitochondria isolated from vitamin E-supplemented (60 mg/kg ip, 3 times/wk for 3 wk) and nonsupplemented mice. Forty male mice were randomly divided into four groups: control + placebo, hypoxia + placebo (H + P), control + vitamin E, and hypoxia + vitamin E. Significant increases in mitochondrial heat shock protein 60 expression and protein carbonyls group levels and decreases in aconitase activity and sulfhydryl group content were found in the H + P group when compared with the control + placebo group. Mitochondrial respiration was significantly impaired in animals from the H + P group, as demonstrated by decreased state 3 respiratory control ratio and ADP-to-oxygen ratio and by increased state 4 with both complex I- and II-linked substrates. Using malate + pyruvate as substrates, hypoxia decreased the respiratory rate in the presence of carbonyl cyanide m-chlorophenylhydrazone and also stimulated oligomycin-inhibited respiration. However, vitamin E treatment attenuated the effect of hypoxia on the mitochondrial levels of heat shock protein 60 and markers of oxidative stress. Vitamin E was also able to prevent most mitochondrial alterations induced by hypobaric hypoxia. In conclusion, hypobaric hypoxia increases mitochondrial oxidative stress while decreasing mitochondrial capacity for oxidative phosphorylation. Vitamin E was an effective preventive agent, which further supports the oxidative character of mitochondrial dysfunction induced by hypoxia.

Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response

Heat stress can negatively affect crop productivity. One way in which plants attempt to alleviate the effects of heat stress is to induce the expression of genes encoding heat shock proteins (HSPs), including small HSPs (sHSPs). We produced transgenic lines of Arabidopsis thaliana expressing a transgene encoding a maize mitochondrial sHSP, ZmHSP22. The transgene, under the control of the cauliflower mosaic virus 35S promoter, is constitutively highly expressed in these lines. As demonstrated by confocal immunofluorescence microscopy and analyses of isolated mitochondria, ZmHSP22 is directed to the mitochondria of Arabidopsis and is processed into the mature form. These transgenic lines demonstrated altered expression of nuclear genes encoding the endogenous mitochondrial sHSP, AtHSP23.6, chloroplast localized AtHSP25.3, class I cytosolic AtHSP17.4, cytosolic AtHSP70‐1 and chloroplast localized AtHSP70‐6, but not cytosolic AtHSP70‐15, following exposure to heat stress. This suggests that the expression of HSPs can be affected by heat‐induced mitochondrial retrograde regulation. Three‐week‐old plants from the transgenic Arabidopsis lines expressing ZmHSP22 have increased thermotolerance, as measured by the maintenance of higher leaf mass following successive days with short periods of heat stress.

JNK2 Translocates to the Mitochondria and Mediates Cytochrome c Release in PC12 Cells in Response to 6-Hydroxydopamine

6-Hydroxydopamine (6-OHDA) causes death of dopaminergic neurons by mitochondrial dysfunction with JNKs as central mediators. Here we provide novel insights into specific actions of JNK isoforms in 6-OHDA-induced death of PC12 cells. Twenty five mum 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus, and at the mitochondria. Inhibition of JNKs by 2 mum SP600125 or transfection with dominant-negative JNK2 (dnJNK2) rescued more than 60% of the otherwise dying PC12 cells after 24 h, whereas transfection with dnJNK1 had no protective effects. In contrast to constitutively present JNK1, JNK2 amounts increased in the nucleus and at the mitochondria after 6-OHDA stimulation. JNK inhibition by SP600125 or transfection of dnJNK2 reduced the pool of active JNKs in the nucleus, the release of cytochrome c, as well as the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase-1. Transfection with dnJNK1, however, had no effects on the translocation of JNKs to the mitochondria or the release of cytochrome c. Our data provide novel functional insights into the pathological role of individual JNK isoforms, the signalosome at the mitochondria, and the mode of JNK-induced release of cytochrome c.

Mortalin is over‐expressed by colorectal adenocarcinomas and correlates with poor survival

Using comparative proteomic analysis we have identified over-expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat-shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild-type tumour suppressor protein p53 and influences the Ras-Raf-MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over-expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over-expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well-defined tumour database.

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.