Toxoplasma gondii Sis1-like J-domain protein is a cytosolic chaperone associated to HSP90/HSP70 complex

Toxoplasma gondii is an obligate intracellular protozoan parasite in which 36 predicted Hsp40 family members were identified by searching the T. gondii genome. The predicted protein sequence from the gene ID TGME49_065310 showed an amino acid sequence and domain structure similar to Saccharomyces cerevisiae Sis1. TgSis1 did not show differences in its expression profile during alkaline stress by microarray analysis. Furthermore, TgSis1 showed to be a cytosolic Hsp40 which co-immunoprecipitated with T. gondii Hsp70 and Hsp90. Structural modeling of the TgSis1 peptide binding fragment revealed structural and electrostatic properties different from the experimental model of human Sis1-like protein (Hdj1). Based on these differences; we propose that TgSis1 may be a potentially attractive drug target for developing a novel anti-T. gondii therapy.     

Assistance for a chaperone: Chlamydomonas HEP2 activates plastidic HSP70B for cochaperone binding

Previous efforts aimed at the biochemical characterization of chloroplast HSP70B were hampered by the observation that recombinant HSP70B was inactive, i.e. incompetent of interacting with its nucleotide exchange factor CGE1. In addition, because heterologously expressed mitochondrial Hsp70 was inactive unless coexpressed with the escort protein Hep1, we wondered whether homologs of Hep1 existed in the chloroplast. Data base searches revealed that algae and higher plants indeed encode at least two HEP homologs, one predicted to be targeted to mitochondria, the others to chloroplasts. Using Chlamydomonas reinhardtii as plant model organism we demonstrate that this alga encodes an HEP homolog (termed HEP2) that is localized to the stroma. HEP2 is expressed constitutively as a low abundance protein with an apparent molecular mass of approximately 21 kDa. In cell extracts HEP2 interacts with HSP70B in an ATP-dependent fashion. Coexpression of HSP70B with HEP2 in Escherichia coli yielded high levels of CGE1-binding competent HSP70B, which also displayed ATPase activity. Inactive HSP70B was more prone to proteolysis than active HSP70B. Although inactive HSP70B interacted with HEP2, it could not be activated. Active HSP70B remained active for 48 h in the absence of HEP2, suggesting that HEP2 was not involved in maintaining HSP70B in an active state. However, some HSP70B expressed as a fusion protein with an N-terminal extension was activated when HEP2 was present during cleavage of the fusion protein, suggesting that in vivo HEP2 might be required for de novo folding of HSP70B after transit peptide cleavage.     

HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii

We report on the molecular and biochemical characterization of HEAT SHOCK PROTEIN 90C (HSP90C), one of the three Hsp90 chaperones encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that HSP90C is a plastidic protein. In the chloroplast, HSP90C was localized to the soluble stroma fraction, but also to thylakoids and low-density membranes containing inner envelopes. HSP90C is expressed under basal conditions and is strongly induced by heat shock and moderately by light. In soluble cell extracts, HSP90C was mainly found to organize into dimers, but also into complexes of high molecular mass. Also, heterologously expressed HSP90C was mainly found in dimers, but tetramers and fewer monomers were detected, as well. HSP90C exhibits a weak ATPase activity with a Km for ATP of approximately 48 microM and a kcat of approximately 0.71 min(-1). This activity was inhibited by the Hsp90-specific inhibitor radicicol. In coimmunoprecipitation experiments, we found that HSP90C interacts with several proteins, among them plastidic HSP70B. The cellular concentration of HSP70B was found to be 2.9 times higher than that of HSP90C, giving a 4.8:1 stoichiometry of HSP70B monomers to HSP90C dimers. The strong inducibility of HSP90C by heat shock implies a role of the chaperone in stress management. Furthermore, its interaction with HSP70B suggests that, similar to their relatives in cytosol and the endoplasmic reticulum, both chaperones might constitute the core of a multichaperone complex involved in the maturation of specific client proteins, e.g. components of signal transduction pathways.     

Role of vesicle-inducing protein in plastids 1 in cpTat transport at the thylakoid

VIPP1 has been shown to be required for the proper formation of thylakoid membranes. However, studies on VIPP1 itself, as well as on PspA, its bacterial homolog, suggests that this protein may be involved in a number of additional functions, including protein translocation. The role of VIPP1 in protein translocation in the chloroplast has not been investigated. To this end, we conducted in vitro thylakoid protein transport assays to look at the effect of VIPP1 on the cpTat pathway, which is one of three translocation pathways found in both the chloroplast and its bacterial progenitor. We found that VIPP1 does indeed enhance protein transport through the cpTat pathway by up to 100%. The VIPP1 effect on cpTat activity occurs without interacting with the substrates or components of the translocon, and does not alter the energy potentials driving this translocation pathway. Instead, VIPP1 greatly enhances the amount of substrate bound productively to the thylakoids. Moreover, the presence of increasing VIPP1 concentrations in the reactions resulted in greater interactions between thylakoid membranes. Taken together, these results demonstrate a stimulatory role for VIPP1 in cpTat transport by enhancement of substrate binding, probably to the membrane lipid regions of the thylakoid. We propose a model in which VIPP1 facilitates reorganization of the thylakoid structure to increase substrate access to productive binding regions of the membrane as an early step in the cpTat pathway.     

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.     

The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila

By screening for mutants exhibiting interactions with a dominant-negative dynamin, we have identified the Drosophila homologue of receptor-mediated endocytosis (Rme) 8, a J-domain-containing protein previously shown to be required for endocytosis in Caenorhabditis elegans. Analysis of Drosophila Rme-8 mutants showed that internalization of Bride of sevenless and the uptake of tracers were blocked. In addition, endosomal organization and the distribution of clathrin were greatly disrupted in Rme-8 cells, suggesting that Rme-8 participates in a clathrin-dependent process. The phenotypes of Rme-8 mutants bear a strong resemblance to those of Hsc70-4, suggesting that these two genes act in a common pathway. Indeed, biochemical and genetic data demonstrated that Rme-8 interacts specifically with Hsc70-4 via its J-domain. Thus, Rme-8 appears to function as an unexpected but critical cochaperone with Hsc70 in endocytosis. Because Hsc70 is known to act in clathrin uncoating along with auxilin, another J-protein, its interaction with Rme-8 indicates that Hsc70 can act with multiple cofactors, possibly explaining its pleiotropic effects on the endocytic pathway.     

Human Fas-associated factor 1 interacts with heat shock protein 70 and negatively regulates chaperone activity

We examined the cell death-inducing property of human Fas-associated factor 1 (hFAF1) in the heat shock signaling pathway. By employing co-immunoprecipitation and peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that hFAF1 binds to the 70-kDa heat shock protein family (Hsc70/Hsp70). Interaction mapping indicated that the 82-180 sequence of hFAF1 directly binds to the N-terminal region containing sequence 1-120 of Hsc70/Hsp70. This binding is very tight regardless of ATP and heat shock treatment. Hsc70/Hsp70 and hFAF1 co-localized in the cytosol and nucleus and concentrated to the perinuclear region by heat shock treatment. We examined how hFAF1 regulates Hsp70 function, and found that hFAF1 inhibited the Hsp70 chaperone activity of refolding denatured protein substrates, accelerated heat shock-induced SAPK/JNK activation, and raised heat shock-induced cell death in a binding dependent manner. These results suggest that hFAF1 prevents cells from recovery after stress by binding to and inhibiting the chaperone activity of Hsp70.     

The chloroplast DnaJ homolog CDJ1 of Chlamydomonas reinhardtii is part of a multichaperone complex containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.     

The molecular chaperone Hsc70 interacts with the vesicular monoamine transporter-2

Synaptic transmission depends on the efficient loading of transmitters into synaptic vesicles by vesicular neurotransmitter transporters. The vesicular monoamine transporter-2 (VMAT2) is essential for loading monoamines into vesicles and maintaining normal neurotransmission. In an effort to understand the regulatory mechanisms associated with VMAT2, we have embarked upon a systematic search for interacting proteins. Glutathione-S-transferase pull-down assays combined with mass spectrometry led to the identification of the 70-kDa heat shock cognate protein (Hsc70) as a VMAT2 interacting protein. Co-immunoprecipitation experiments in brain tissue and heterologous cells confirmed this interaction. A direct binding was observed between the amino terminus and the third cytoplasmic loop of VMAT2, as well as, a region containing the substrate binding and the carboxy-terminal domains of Hsc70. Furthermore, VMAT2 and Hsc70 co-fractionated with purified synaptic vesicles obtained from a sucrose gradient, suggesting that this interaction occurs at the synaptic vesicle membrane. The functional significance of this novel VMAT2/Hsc70 interaction was examined by performing vesicular uptake assays in heterologous cells and purified synaptic vesicles from brain tissue. Recombinant Hsc70 produced a dose-dependent inhibition of VMAT2 activity. This effect was mimicked by the closely related Hsp70 protein. In contrast, VMAT2 activity was not altered in the presence of previously denatured Hsc70 or Hsp70, as well as the unrelated Hsp60 protein; confirming the specificity of the Hsc70 effect. Finally, a purified Hsc70 fragment that binds VMAT2 was sufficient to inhibit VMAT2 activity in synaptic vesicles. Our results suggest an important role for Hsc70 in VMAT2 function and regulation.     

Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1

The chaperone activity of Hsp70 is influenced by the activities of both positive and negative regulatory proteins. In this study, we provide first time evidence for the stimulating effect of the Hsp70-interacting protein Hip on the chaperone activity in the mammalian cytosol. Overexpressing Hip enhances the refolding of the heat-inactivated reporter enzyme luciferase expressed in hamster lung fibroblasts. Also, it protects luciferase from irreversible denaturation under conditions of ATP depletion. We demonstrate that these stimulating actions depend on both the presence of the central Hsp70-binding site and the amino-terminal homo-oligomerization domain of Hip. The carboxyl terminus (amino acids 257-368) comprising the 7 GGMP repeats (Hsc70-like domain) and the Sti1p-like domain are dispensable for the Hip-mediated stimulation of the cellular chaperone activity. Bag-1, which inhibits the Hsp70 chaperone activity both in vitro and in vivo, was found to compete with the stimulatory action of Hip. In cells overexpressing both Hip and Bag-1, the inhibitory effects of Bag-1 were found to be dominant. Our results reveal that in vivo a complex level of regulation of the cellular chaperone activity exists that not only depends on the concentration of Hsp70 but also on the concentration, affinity, and intracellular localization of positive and negative coregulators. As the Hsp70 chaperone machine is also protective in the absence of ATP, our data also demonstrate that cycling between an ATP/ADP-bound state is not absolutely required for the Hsp70 chaperone machine to be active in vivo.     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFα‐induced signalling pathway have not been fully elucidated. We report here that HSP70 over‐expression in human colon cancer cells can inhibit TNFα‐induced NFκB activation but promote TNFα‐induced activation of c‐Jun N‐terminal kinase (JNK) through interaction with TNF receptor (TNFR)‐associated factor 2 (TRAF2). We provide evidence that HSP70 over‐expression can sequester TRAF2 in detergent‐soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor‐interacting protein (RIP1) and IκBα kinase (IKK) signalosome to the TNFR1–TRADD complex and inhibited NFκB activation after TNFα stimuli. In addition, we found that HSP70–TRAF2 interaction can promote TNFα‐induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFα‐induced activation of NFκB and JNK through interaction with TRAF2, contributing to the pro‐apoptotic roles of HSP70 in TNFα‐induced apoptosis of human colon cancer cells.     

Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen

Hsp47 is a heat-shock protein that interacts transiently with procollagen during its folding, assembly and transport from the endoplasmic reticulum (ER) of mammalian cells. It has been suggested to carry out a diverse range of functions, such as acting as a molecular chaperone facilitating the folding and assembly of procollagen molecules, retaining unfolded molecules within the ER, and assisting the transport of correctly folded molecules from the ER to the Golgi apparatus. Here we define the substrate recognition of Hsp47, demonstrating that it interacts preferentially with triple-helical procollagen molecules. The association of Hsp47 with procollagen coincides with the formation of a collagen triple helix. This demonstrates that Hsp47's role in procollagen folding and assembly is distinct from that of prolyl 4-hydroxylase. These results indicate that Hsp47 acts as a novel molecular chaperone, potentially stabilizing the correctly folded collagen helix from heat denaturation before its transport from the ER.     

The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation

The J-domain co-chaperones work together with the heat shock protein 70 (HSP70) chaperone to regulate many cellular events, but the mechanism underlying the J-domain-mediated HSP70 function remains elusive. We studied the interaction between human-inducible HSP70 and Homo sapiens J-domain protein (HSJ1a), a J domain and UIM motif-containing co-chaperone. The J domain of HSJ1a shares a conserved structure with other J domains from both eukaryotic and prokaryotic species, and it mediates the interaction with and the ATPase cycle of HSP70. Our in vitro study corroborates that the N terminus of HSP70 including the ATPase domain and the substrate-binding β-subdomain is not sufficient to bind with the J domain of HSJ1a. The C-terminal helical α-subdomain of HSP70, which was considered to function as a lid of the substrate-binding domain, is crucial for binding with the J domain of HSJ1a and stimulating the ATPase activity of HSP70. These fluctuating helices are likely to contribute to a proper conformation of HSP70 for J-domain binding other than directly bind with the J domain. Our findings provide an alternative mechanism of allosteric activation for functional regulation of HSP70 by its J-domain co-chaperones.     

NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease

When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the alpha-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC.ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process.     

The rough endoplasmic reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and B?chinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.