EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

Background

The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods

Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results

The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions

Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.     

Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR

The most common mutation ofthe cystic fibrosis transmembrane conductance regulator(CFTR), F508, is a trafficking mutant that has prolongedassociations with molecular chaperones and is rapidly degraded, atleast in part by the ubiquitin-proteasome system. Sodium4-phenylbutyrate (4PBA) improves F508-CFTR trafficking and functionin vitro in cystic fibrosis epithelial cells and in vivo. To furtherunderstand the mechanism of action of 4PBA, we tested the hypothesisthat 4PBA modulates the targeting of F508-CFTR for ubiquitinationand degradation by reducing the expression of Hsc70 in cystic fibrosisepithelial cells. IB3-1 cells (genotype F508/W1282X) that weretreated with 0.05-5 mM 4PBA for 2 days in culture demonstrated adose-dependent reduction in Hsc70 protein immunoreactivity and mRNAlevels. Immunoprecipitation with Hsc70-specific antiserum demonstratedthat Hsc70 and CFTR associated under control conditions and thattreatment with 4PBA reduced these complexes. Levels of immunoreactiveHsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBAtreatment. These data suggest that 4PBA may improve F508-CFTRtrafficking by allowing a greater proportion of mutant CFTR to escapeassociation with Hsc70.

     

In vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress

Hsp70 molecular chaperones have been shown to play an important role in helping cells to cope with adverse environments, especially in response to high temperatures. The molecular chaperone function of Hsc70 at low temperature was investigated. A cold-inducible spinach cytosolic Hsc70 was subcloned into a protein expression vector and the recombinant protein was expressed in bacterial cells. Recombinant Hsc70 bound a permanently unfolded substrate: alpha-carboxymethylated lactalbumin (CMLA) in the presence of 3 mM ATP and MgCl(2) at low temperature (4 and -4 degrees C). Radiolabeling with (35)S-Met and (35)S-Cys and immunoprecipitation with cytosolic Hsc70 monoclonal antibodies showed that there were several proteins co-immunoprecipitated at low temperature (4 and -4 degrees C) but not at room temperature. Enhanced co-purification of sHsp17.7 with Hsc70 at low temperature was observed and suggests that co-chaperone interactions can contribute to molecular chaperone function during cold stress. These results suggest that the molecular chaperone Hsc70 may have a functional role in plants during low temperature stress.     

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C-terminus of Hsc70-interacting protein (CHIP) with a U-box domain is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U-box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C-terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self-ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality-control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.     

Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma

In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins, called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological processes including life/death cell balance and stress response. The role of Stv has been poorly studied in the context of abiotic stress and particularly during and after cold stress. In this study we investigated the temporal expression of Stv gene and protein in adult flies during both the cold stress (up to 9 h at 0 °C) and the subsequent recovery phase (up to 8 h at 25 °C). Because BAG proteins can regulate positively and negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70 and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during the cold period followed by a significant up-regulation during the recovery period. A coordinated response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our findings indicate that Stv expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our results support the suggestion that Stv and human BAG3 may be functional homologs.     

PKCι counteracts oxidative stress by regulating Hsc70 in an esophageal cancer cell line

Using a glutathione S-transferase pull-down liquid chromatography–coupled tandem mass spectrometry approach and immunoprecipitation/immunoblot analysis, we found that heat shock cognate protein 70 (Hsc70) was involved in the complex formed by atypical protein kinase Cι (PKCι) and LC3 in the esophageal cancer cell line KYSE30. Further study indicated that Hsc70 was targeted by autophagic degradation, and knockdown of PKCι down-regulated Hsc70 by promoting autophagy. PKCι knockdown sensitized cells to oxidative stress-induced apoptosis, whereas forced PKCι expression counteracted the oxidative stress-induced apoptosis via Hsc70.     

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.     

Heat shock cognate protein 70 contributes to <Emphasis Type="Italic">Brucella</Emphasis> invasion into trophoblast giant cells that cause infectious abortion

Background  

The cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG) cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells.     

A plant reovirus hijacks the DNAJB12–Hsc70 chaperone complex to promote viral spread in its planthopper vector

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus-encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black-streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus-containing tubules composed of nonstructural membrane protein P7-1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER-associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co-chaperone Hsc70, are activated by SRBSDV to facilitate ER-to-cytosol export of P7-1 tubules in S. furcifera. Both P7-1 of SRBSDV and Hsc70 directly bind to the J-domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7-1, but Hsc70 overexpression promotes the transport of P7-1 from the ER to the cytosol to initiate tubule assembly. Thus, P7-1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7-1 tubule assembly, suggesting that the proper folding and assembly of P7-1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7-1 tubules in virus-infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7-1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER-to-cytosol transport of P7-1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.     

Heat Shock Protein Cognate 70-4 and an E3 Ubiquitin Ligase,CHIP, Mediate Plastid-Destined Precursor Degradation through the Ubiquitin-26S Proteasome System in Arabidopsis

Plastid-targeted proteins pass through the cytosol as unfolded precursors. If proteins accumulate in the cytosol, they can form nonspecific aggregates that cause severe cellular damage. Here, we demonstrate that high levels of plastid precursors are degraded through the ubiquitin-proteasome system (UPS) in Arabidopsis thaliana cells. The cytosolic heat shock protein cognate 70-4 (Hsc70-4) and E3 ligase carboxy terminus of Hsc70-interacting protein (CHIP) were highly induced in plastid protein import2 plants, which had a T-DNA insertion at Toc159 and showed an albino phenotype and a severe defect in protein import into chloroplasts. Hsc70-4 and CHIP together mediated plastid precursor degradation when import-defective chloroplast-targeted reporter proteins were transiently expressed in protoplasts. Hsc70-4 recognized specific sequence motifs in transit peptides and thereby led to precursor degradation through the UPS. CHIP, which interacted with Hsc70-4, functioned as an E3 ligase in the Hsc70-4–mediated protein degradation. The physiological role of Hsc70-4 was confirmed by analyzing Hsc70-4 RNA interfernce plants in an hsc70-1 mutant background. Plants with lower Hsc70 levels exhibited abnormal embryogenesis, resulting in defective seedlings that displayed high levels of reactive oxygen species and monoubiquitinated Lhcb4 precursors. We propose that Hsc70-4 and CHIP mediate plastid-destined precursor degradation to prevent cytosolic precursor accumulation and thereby play a critical role in embryogenesis.     

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.     

A novel receptor – ligand pathway for entry of <Emphasis Type="Italic">Francisella tularensis</Emphasis> in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

Background  

Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.     

Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

Background

Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet.

Results

Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca²⁺ uptake. Ca²⁺-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca²⁺ release from the endoplasmic reticulum via both Ins₃P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca²⁺uptake.

Conclusions

Our data indicate that TG2 might act as a Ca²⁺ sensor to amplify endoplasmic reticulum-derived Ca²⁺ signals to enhance mitochondria Ca²⁺ uptake. Since enhanced mitochondrial Ca²⁺ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca²⁺ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca²⁺ homeostasis.     

Autophosphorylation of the pea mitochondrial heat-shock protein homolog

Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [γ-³²P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.     

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

Background  

The zinc uptake regulator Zur is a Zn²⁺-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis.     

Constitutive heat shock protein 70 interacts with α-enolase and protects cardiomyocytes against oxidative stress

Abstract

Constitutive heat shock protein 70 (Hsc70) is a molecular chaperone that has been shown to protect cardiomyocytes against oxidative stress. However, the molecular mechanism responsible for this protection remains uncertain. To understand the mechanism associated with the myocardial protective role of Hsc70, we have embarked upon a systematic search for Hsc70-interacting proteins. Using adenosine diphosphate (ADP) affinity chromatography and mass spectrometry, we have identified α-enolase, a rate-limiting enzyme in glycolysis, as a novel Hsc70-interacting protein in the myocardium of both sham and myocardial ischemia-reperfused Sprague–Dawley rat hearts. This interaction was confirmed by co-immunoprecipitation (IP) assays in the myocardial tissues and H9c2 cardiomyocytes and protein overlay assay (POA). It was further shown that Hsc70-overexpression alleviated the H₂O₂-induced decrease of α-enolase activity and cell damage, and Hsc70 deficiency aggravated the decrease of α-enolase activity and cell damage in H₂O₂ treated H9c2 cells. Our research suggests that the protective effect of Hsc70 on the cardiomyocytes against oxidative stress is partly associated with its interaction with α-enolase.     

Geldanamycin induces production of heat shock protein 70 and partially attenuates ototoxicity caused by gentamicin in the organ of Corti explants

Background  

Heat shock protein 70 (HSP70) protects inner ear cells from damage and death induced by e.g. heat or toxins. Benzoquinone ansamycin antibiotic geldanamycin (GA) was demonstrated to induce the expression of HSP70 in various animal cell types. The aim of our study was to investigate whether GA induces HSP70 in the organ of Corti (OC), which contains the auditory sensory cells, and whether GA can protect these cells from toxicity caused by a common aminoglycoside antibiotic gentamicin.     

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

Background  

In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.     

Heat shock cognate protein 70 controls Borna disease virus replication via interaction with the viral non-structural protein X

Borna disease virus (BDV) is a non-segmented, negative-sense RNA virus and has the property of persistently infecting the cell nucleus. BDV encodes a 10-kDa non-structural protein, X, which is a negative regulator of viral polymerase activity but is essential for virus propagation. Recently, we have demonstrated that interaction of X with the viral polymerase cofactor, phosphoprotein (P), facilitates translocation of P from the nucleus to the cytoplasm. However, the mechanism by which the intracellular localization of X is controlled remains unclear. In this report, we demonstrate that BDV X interacts with the 71 kDa molecular chaperon protein, Hsc70. Immunoprecipitation assays revealed that Hsc70 associates with the same region of X as P and, interestingly, that expression of P interferes competitively with the interaction between X and Hsc70. A heat shock experiment revealed that BDV X translocates into the nucleus, dependent upon the nuclear accumulation of Hsc70. Furthermore, we show that knockdown of Hsc70 by short interfering RNA decreases the nuclear localization of both X and P and markedly reduces the expression of viral genomic RNA in persistently infected cells. These data indicate that Hsc70 may be involved in viral replication by regulating the intracellular distribution of X.     

Calcium Ionophore-Induced Degradation of Neurofilament and Cell Death in MSN Neuroblastoma Cells

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca²⁺-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EQTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.