Chaperone Regulator Research Articles

Neuroprotective Function of DJ-1 in Parkinson’s Disease

Parkinson's disease (PD) is caused by dopaminergic neuronal death in the substantia nigra, resulting in a reduced level of dopamine in the striatum. Oxidative stress and mitochondrial dysfunction are thought to be major causes of neurodegeneration in PD. Although genetic and environmental factors are thought to affect the onset of PD, precise mechanisms at the molecular level have not been elucidated. The DJ-1 gene is a causative gene for familial PD (park7) and also an oncogene. DJ-1 has various functions, including transcriptional regulation, antioxidative stress reaction, and chaperone, protease, and mitochondrial regulation, and its activity is regulated by its oxidative status, especially that of cysteine 106 (C106) of DJ-1. Excess oxidation of DJ-1, which renders DJ-1 inactive, has been observed in patients with sporadic PD and Alzheimer's disease, suggesting that DJ-1 also participates in the onset and pathogenesis of sporadic PD as well as familial PD. DJ-1 is also a stress sensor and its expression is increased upon various stresses, including oxidative stress. In this review, we describe functions of DJ-1 against oxidative stress and possible roles of DJ-1 in the pathogenesis of PD.

Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones

Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the β sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the β sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the β sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.

MiR-30d, miR-181a and miR-199a-5p cooperatively suppress the endoplasmic reticulum chaperone and signaling regulator GRP78 in cancer

GRP78, a major endoplasmic reticulum chaperone and signaling regulator, is commonly overexpressed in cancer. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemoresistance to cancer, thereby contributing to tumorigenesis. Thus, therapies aimed at decreasing GRP78 levels, which results in the inhibition of tumor cell proliferation and resensitization of tumor cells to chemotherapeutic drugs may hold promise for cancer treatment. Despite advances in our understanding of GRP78 actions, little is known about endogenous inhibitors controlling its expression. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78, under the hypothesis that these miRNAs may serve as therapeutic agents. Here, we report that three miRNAs (miR-30d, miR-181a, miR-199a-5p) predicted to target GRP78 are down-regulated in prostate, colon and bladder tumors, and human cancer cell lines. We show that in C42B prostate cancer cells, these miRNAs down-regulate GRP78 and induce apoptosis by directly targeting its 3' untranslated region. Importantly, we demonstrate that the three miRNAs act cooperatively to decrease GRP78 levels, suggesting that multiple miRNAs may be required to efficiently control the expression of some genes. In addition, delivery of multiple miRNAs by either transient transfection or lentivirus transduction increased the sensitivity of cancer cells to the histone deacetylase inhibitor, trichostatin A, in C42B, HCT116 and HL-60 cells. Together, our results indicate that the delivery of co-transcribed miRNAs can efficiently suppress GRP78 levels and GRP78-mediated chemoresistance, and suggest that this strategy holds therapeutic potential.

The Absence of Heat Shock Protein HSP101 Affects the Proteome of Mature and Germinating Maize Embryos

Maize heat shock protein HSP101 accumulates during embryo maturation and desiccation and persists at high levels during the first 24 h following kernel imbibition in the absence of heat stress. This protein has a known function in disaggregation of high molecular weight complexes and has been proposed to be a translational regulator of specific mRNAs. Here, a global proteomic approach was used to identify changes in the maize proteome due to the absence of HSP101 in embryos from mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from the mature dry embryo exhibited statistically significant expression changes in the L10 inbred hsp101 mutant (hsp101-m5::Mu1/hsp101-m5::Mu1) line as compared to the corresponding wild type (Hsp101/Hsp101). Additional six spots reproducibly showed qualitative changes between the mutant and wild-type mature and germinating embryos. Several chaperones, translation-related proteins, actin, and enzymes participating in cytokinin metabolism were identified in these spots by tandem mass-spectrometry (MS). The proteomic changes partially explain the altered root growth and architecture observed in young hsp101 mutant seedlings. In addition, specific protein de novo synthesis was altered in the 24 h-imbibed mutant embryos indicating that maize HSP101 functions as both chaperone and translational regulator during germination. Supporting this, HSP101 was found as part of Cap-binding and translation initiation complexes during early kernel imbibition. Overall, these findings expose the relevance of maize HSP101 for protein synthesis and balance mechanisms during germination.

Abstract 2773: HSP90 inhibitors target KRAS mutant human tumors through degradation of STK33

Abstract Mutations in the KRAS proto-oncogene occur in ∼30% of human cancers and are particularly prevalent in adenocarcinomas of the pancreas, lung, and colon. Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS have not met with success. We have recently shown that cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, supporting STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33, which remain to be elucidated, are not yet available. Using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex and BAG2, a member of the BAG family of molecular chaperone regulators, bind to and stabilize STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon cancer-initiating cells harboring mutant KRAS. Similar to HSP90 inhibition, short hairpin RNA-mediated suppression of BAG2 depleted STK33, whereas BAG2 overexpression rescued STK33 protein levels in the presence of HSP90 inhibitors, resulting in diminished apoptosis in KRAS mutant cancer cells. These findings (1) provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, (2) indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, (3) identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential, and (4) point to BAG2 as a candidate cancer drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2773. doi:1538-7445.AM2012-2773

Abstract A8: Defining specific microRNAs that cooperatively regulate GRP78, an ER chaperone and signaling regulator, in cancer

Abstract GRP78, a major endoplasmic reticulum (ER) chaperone and signaling regulator, is commonly over-expressed in cancer and contributes to cancer cell proliferation, survival and tumorigenesis. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemo-resistance to cancer. Thus, targeting GRP78 to inhibit tumor survival and proliferation, sensitize tumor cells to chemotherapeutic drugs and consequently induce apoptosis serves as a promising avenue in anti-cancer therapy. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78. We report that multiple miRNAs (miR-30d, miR-181a, miR-199-5p) commonly down-regulated in human cancer cell lines can repress GRP78 and lead to increased apoptosis in C42B prostate cancer cells. Luciferase reporter assays indicate that these miRNAs act as negative regulators of GRP78 by directly targeting the 3′ untranslated region of GRP78. Importantly, the combination of multiple miRNAs cooperated resulting in greater inhibition on GRP78 than individual miRNAs, suggesting the efficacy of tumor suppression by combined miRNAs. In addition, we showed that transfection of the multiple miRNAs in C42B cells increased sensitivity of cancer cells to trichostatin A (TSA), a histone deacetylase inhibitor, induced apoptosis, and inhibited cell proliferation and colony formation. Therefore, we suggest that delivery of combinations of specific miRNAs could be a novel anti-cancer approach capable of suppressing GRP78 and in turn inhibiting GRP78-mediated tumorigenesis. Citation Format: Sheng-Fang Su, Amy S. Lee, Gangning Liang. Defining specific microRNAs that cooperatively regulate GRP78, an ER chaperone and signaling regulator, in cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A8.

Proteomic methods unravel the protein quality control in Escherichia coli

Protein quality control is an essential process in all living organisms. A network of folding helper proteins and proteases ushers proteins into their native conformation, safeguards their structure under adverse environmental conditions, and, if all else fails, degrades proteins at the end of their life time. Escherichia coli is a versatile model organism used in the analysis of fundamental cellular processes. Much of what we know about protein quality control has been discovered in this microorganism. In the investigation of the mode of action, regulation and substrate specificity of chaperones, thiol-disulfide isomerases and proteases, proteomic methods have been playing a key role. Here, we provide a condensed overview about the protein quality control network in E. coli and the remarkable contributions of proteomics to our current knowledge.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

BackgroundThe expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach.ResultsOverexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response.ConclusionsInefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity

The DNL-type zinc-finger protein DNLZ regulates the activity and solubility of the human mitochondrial chaperone HSPA9. To identify DNLZ residues that are critical for chaperone regulation, we carried out an alanine mutagenesis scan of charged residues in a W115I mutant of human DNLZ and assessed the effect of each mutation on interactions with HSPA9. All mutants analyzed promote the solubility of HSPA9 upon expression in Escherichia coli. However, binding studies examining the effect of DNLZ mutants on chaperone tryptophan fluorescence identified three mutations (R81A, H107A, and D111A) that decrease DNLZ binding affinity for nucleotide-free chaperone. In addition, ATPase measurements revealed that DNLZ–R81A and DNLZ–D111A both stimulate the catalytic activity HSPA9, whereas DNLZ–H107A does not elicit an increase in activity even when present at a concentration that is 10-fold higher than the level required for half-maximal stimulation by DNLZ. These findings implicate a conserved histidine as critical for DNLZ regulation of mitochondrial HSPA9 catalytic activity.

Abstract SY06-01: Critical role of the stress chaperone and cell signaling regulator GRP78 in cancer

Abstract SY06-01: Critical role of the stress chaperone and cell signaling regulator GRP78 in cancer

Differential regulation of redox proteins and chaperones in HbEβ‐thalassemia erythrocyte proteome

In (hemoglobin, Hb) HbEβ-thalassemia, HbE (β-26 Glu→Lys) interacts with β-thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ-thalassemic patients compared to normal. We have used 2-DE and MALDI-MS/MS-based techniques to investigate the differential proteome profiling of membrane and Hb-depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ-thalassemia patients and normal volunteers. Our study showed that redox regulators such as peroxiredoxin 2, Cu-Zn superoxide dismutase and thioredoxin and chaperones such as α-hemoglobin stabilizing protein and HSP-70 were upregulated in HbEβ-thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin-based junctional complex in the membrane skeleton of HbEβ-thalassemic erythrocytes upon detection of low molecular weight fragments of β-spectrin and decrease in β-actin and dematin content. We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.

BAG-1 enhances cell–cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell–cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell–cell adhesion and suggest an important function in epidermal cohesion.

Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix

Calreticulin (CRT), a chaperone and Ca(2+) regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts deficient in CRT (CRT(-/-) MEFs) have reduced transcript levels of fibrillar collagen I and III and less soluble collagen as compared with wild type MEFs. Correspondingly, fibroblasts engineered to overexpress CRT have increased collagen type I transcript and protein. Collagen expression appears to be regulated by endoplasmic reticulum (ER) calcium levels and intracellular CRT, because thapsigargin treatment reduced collagen expression, whereas addition of exogenous recombinant CRT had no effect. CRT(-/-) MEFs exhibited increased ER retention of collagen, and collagen and CRT were co-immunoprecipitated from isolated cell lysates, suggesting that CRT is important for trafficking of collagen through the ER. CRT(-/-) MEFs also have reduced type I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). Here we report the creation of a novel mouse model where GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in the Purkinje cells (PCs). GRP78 depleted PCs activate UPR including induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2α phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding where abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78 null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

Chaperone-protease systems in regulation and protein quality control in Bacillus subtilis

The Gram-positive model organism Bacillus subtilis is extremely well adapted to changing environmental conditions. The chaperone-protease ClpCP and other AAA+ proteases constitute an important component of the B. subtilis protein quality control system that is essential for survival during stress. In this review, we discuss recent discoveries concerning the molecular mechanism, regulation and localization of proteases and chaperones in B. subtilis.

Hsp70 and cardiac surgery: molecular chaperone and inflammatory regulator with compartmentalized effects

Open heart surgery is a unique model to study the interplay between cellular injury, regulation of inflammatory responses and tissue repair. Stress-inducible heat shock protein 70-kDa (Hsp70) provides a molecular link between these events. In addition to molecular chaperoning, Hsp70 exerts modulatory effects on endothelial cells and leukocytes involved in inflammatory networks. Hsp70 residing in the intracellular compartment is part of an inhibitory feedback loop that acts on nuclear factor kappaB (NF-kappaB). In contrast, extracellular Hsp70 is recognized by multiple germline-encoded immune receptors, e.g., Toll-like receptor (TLR) 2, TLR4, LOX-1, CD91, CD94, CCR5 and CD40. Hsp70 is thereby able to enhance chemotaxis, phagocytosis and cytolytic activity of innate immune cells and stimulate antigen-specific responses. These apparent contradictory pro- and anti-inflammatory effects of endogenous Hsp70 in the context of cardiac surgery are still not fully understood. An all-embracing model of the compartmentalized effects of endogenous Hsp70 in the orchestration of inflammatory responses in cardiac surgery is proposed.

Endoplasmic Reticulum (ER) Chaperone Regulation and Survival of Cells Compensating for Deficiency in the ER Stress Response Kinase, PERK

The activity of PERK, an endoplasmic reticulum (ER) transmembrane protein kinase, assists in an ER stress response designed to inhibit general protein synthesis while allowing upregulated synthesis of selective proteins such as the ATF4 transcription factor. PERK null mice exhibit phenotypes that especially affect secretory cell types. Although embryonic fibroblasts from these mice are difficult to transfect with high efficiency, we have generated 293 cells stably expressing the PERK-K618A dominant negative mutant. 293/PERK-K618A cells, in response to ER stress: (a) do not properly inhibit general protein synthesis, (b) exhibit defective/delayed induction of ATF4 and BiP, and (c) exhibit exuberant splice activation of XBP1 and robust cleavage activation of ATF6, with abnormal regulation of calreticulin levels. The data suggest compensatory mechanisms allowing for cell survival in the absence of functional PERK. Interestingly, although induction of CHOP (a transcription factor implicated in apoptosis) is notably delayed after onset of ER stress, 293/PERK-K618A cells eventually produce CHOP at normal or even supranormal levels and exhibit increased apoptosis either in response to general ER stress or, more importantly, to specific misfolded secretory proteins.

The BAG proteins: a ubiquitous family of chaperone regulators.

The BAG (Bcl-2 associated athanogene) family is a multifunctional group of proteins that perform diverse functions ranging from apoptosis to tumorigenesis. An evolutionarily conserved group, these proteins are distinguished by a common conserved region known as the BAG domain. BAG genes have been found in yeasts, plants, and animals, and are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in carcinogenesis, HIV infection, and Parkinson's disease. These proteins are therefore potential therapeutic targets, and their expression in cells may serve as a predictive tool for such diseases. In plants, the Arabidopsis thaliana genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. Three members contain a calmodulin-binding domain possibly reflecting differences between plant and animal programmed cell death. This review summarizes current understanding of BAG proteins in both animals and plants.

Modulation of the hippocampal protein machinery in voluntary and treadmill exercising rats

Information about protein expression studies in the brain of exercising and sedentary animals is limited. Cognitive functions change during exercise and the aim of this study was to investigate rat protein levels of the protein machinery in the hippocampus, the main cognitive brain area for spatial learning and memory, in exercising rats. Protein fluctuations may reflect functional variation during exercise. Male Sprague–Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats undertaking voluntary exercise from 5 months to 23 months and the third undertaking involuntary exercise on a treadmill from 5 months to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification assigning spots to proteins and determination of coomassie-densities was carried out. Heterogeneous nuclear ribonucleoprotein K, one protein variant of heat shock cognate 71 kDa protein and BAG family molecular chaperone regulator 5 showed differential protein levels in the three groups when a p-value of < 0.005 was considered as statistically significant thus respecting multiple testing. The biological meaning of changed protein levels in hippocampus under different conditions of exercise is not known but warrants further investigation.

Trypanosoma brucei bruceiinduces alteration in the head proteome of the tsetse fly vectorGlossina palpalis gambiensis

Parasitic manipulations of host behaviour are known from a wide range of host-parasite associations. However, the understanding of these phenomena is far from complete and detailed investigation of their proximate causes is needed. Many studies report behavioural modifications, such as altered feeding rates in tsetse fly (Glossina) infected with the mature transmissible stage (i.e. metacyclic) of the trypanosomes. Here, bidimensional (2D) gel electrophoresis and mass spectrometry were employed to analyse and compare the head proteome between four Glossina palpalis gambiensis categories (uninfected, refractory, mature infection, immature infection). Twenty-four protein spots specifically present or absent in the head of metacyclic-infected flies were observed. These protein spots were subsequently identified and functionally classified as glycolitic, neurotransmiter synthesis, signalling, molecular chaperone and transcriptional regulation proteins. Our results indicate altered energy metabolism in the head of metacyclic-infected tsetse flies. Some of the proteins identified, such as casein kinase 2 and jun kinase have previously been shown to play critical roles in apoptosis in insect neurones. In addition, we found two pyridoxal-dependent decarboxylases (dopa decarboxylase and alpha methyldopa hypersensitive protein), suggesting a modification of serotonin and/or dopamine in the brain of metacyclic-infected tsetse flies. Our data pave the way for future investigation of the alteration of the glossina central nervous system during infection by trypanosomes.