Hsp70 Isoforms Research Articles

The yeast stress inducible Ssa Hsp70 reduces α-synuclein toxicity by promoting its degradation through autophagy.

The mechanism underlying the role of Hsp70s in toxicity associated with intracellular accumulation of toxic protein inclusions is under intense investigation. In current study, we examined the roles of all different isoforms of yeast cytosolic Ssa Hsp70 on α-synuclein mediated cellular toxicity. The study showed that yeast cells expressing stress-inducible Ssa3 or Ssa4 as sole Ssa Hsp70 isoforms, reduced α-synuclein toxicity better than those expressing a constitutive counterpart. The protective effect of stress-inducible Ssa Hsp70s was not α-syn specific, but more general to other inclusion forming proteins such as polyQ. We show that the protective effect is not by induction of a general stress response in Ssa3 cells rather by promoting α-synuclein degradation through autophagy. The present study revealed that effect of Hsp70s was isoform dependent, and that autophagy protects Ssa3 cells from the deleterious effects of toxic protein inclusions.

Hsp90 inhibition in adrenocortical carcinoma: Limited drug synergism with mitotane

90 kDa heat shock proteins (Hsp90) act as protein chaperones and play a role in modulating endoplasmic reticulum (ER) stress. Hsp90 inhibitors are under clinical investigation as cancer treatment. Mitotane therapy of adrenocortical carcinoma (ACC) has been shown to act through lipid-induced ER-stress. To explore the potential of Hsp90 inhibitors in ACC as a single agent and in combination with mitotane, we analyzed two independent gene expression data sets of adrenal tumors in silico and treated the ACC cell line model NCI-H295 with Hsp90 inhibitors BIIB021 (B) and CCT18159 (C) alone and in combination with mitotane. ER-stress markers were monitored by immunoblotting. Drug synergism was quantified using the median effect model with cell viability as read-out. Cytosolic Hsp90 isoforms AA1 and AB1 were significantly overexpressed in ACC. Viability of H295 cells was impaired by B and C as single agents with an EC50 of 5.7 × 10−6M and 12.1 × 10−6M. B but not C dose-dependently increased XBP1 splicing and CHOP expression indicative of ER-stress activation. ER-stress marker expression was enhanced by co-incubation of B with 10 μM but not 5 μM mitotane. Maximal CHOP expression was induced by 25 μM mitotane alone with no additional effect of B. Combination indices (CI) of B and C with mitotane ranged from 0.64 to 1.38 and 0.68 to 1.30, respectively where CI values < 0.5 support clinically-relevant drug synergism. In conclusion, Hsp90 paralogs are differentially expressed in ACC and B but not C activates ER-stress in ACC cells. No meaningful drug synergism of Hsp90 inhibitors with mitotane was observed.

Engineered Hsp70 chaperones prevent A\u03b242-induced memory impairments in a Drosophila model of Alzheimer\u2019s disease

Proteinopathies constitute a group of diseases in which certain proteins are abnormally folded leading to aggregation and eventual cell failure. Most neurodegenerative diseases belong to protein misfolding disorders and, among them, Alzheimer’s disease (AD) is the most prevalent. AD is characterized by accumulation of the amyloid-β42 (Aβ42) peptide in the extracellular space. Hence, we genetically engineered a molecular chaperone that was selectively delivered to this cellular location. It has been reported that the heat shock protein 70 (Hsp70) binds Aβ42 preventing self-aggregation. Here, we employed two isoforms of the Hsp70, cytosolic and extracellular, to evaluate their potential protective effect against the memory decline triggered by extracellular deposition of Aβ42. Both Hsp70 isoforms significantly improved memory performance of flies expressing Aβ42, irrespective of their age or the level of Aβ42 load. Using olfactory classical conditioning, we established a Drosophila model of AD based on Aβ42 neurotoxicity and monitored memory decline through aging. The onset of the memory impairment observed was proportional to the cumulative level of Aβ42 in the Drosophila brain. These data support the use of this Drosophila model of AD to further investigate molecules with a protective activity against Aβ42-induced memory loss, contributing to the development of palliative therapies for AD.

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's-related Tau protein

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90β in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90β, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90β in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90β have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ββ homodimers and no GR•Hsp90αβ heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αβ heterodimers. The formation of GR•Hsp90αβ heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αβ heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

A secreted Heat shock protein 90 of Trichomonas vaginalis.

Trichomonas vaginalis is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since Trichomonas Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.

Ectromelia virus upregulates the expression of heat shock protein70 to promote viral replication.

The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcription‑quantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member1B (Hspa1b), was highly upregulated during ECTV infection invitro and invivo. Subsequently, overexpression of Hspa1b protein and small interfering RNA‑mediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.

Characterizing functional differences in sea anemone Hsp70 isoforms using budding yeast

Marine organisms experience abiotic stressors such as fluctuations in temperature, UV radiation, salinity, and oxygen concentration. Heat shock proteins (HSPs) assist in the response of cells to these stressors by refolding and maintaining the activity of damaged proteins. The well-conserved Hsp70 chaperone family is essential for cell viability as well as the response to stress. Organisms possess a variety of Hsp70 isoforms that differ slightly in amino acid sequence, yet very little is known about their functional relevance. In this study, we undertook analysis of three principal Hsp70 isoforms NvHsp70A, B, and D from the starlet sea anemone Nematostella vectensis. The functionality of Hsp70 isoforms in the starlet sea anemone was assessed through transcriptional analysis and by heterologous expression in budding yeast Saccharomyces cerevisiae. Interestingly, these isoforms were found to not only differ in expression under stress but also appear to have functional differences in their ability to mediate the cellular stress program. These results contribute to an understanding of Hsp70 isoform specificity, their shared and unique roles in response to acute and chronic environmental stress, and the potential basis of local adaptation in populations of N. vectensis.

Dynamics-based community analysis and perturbation response scanning of allosteric interaction networks in the TRAP1 chaperone structures dissect molecular linkage between conformational asymmetry and sequential ATP hydrolysis

Allosteric interactions of the Hsp90 chaperones with cochaperones and diverse protein clients can often exhibit distinct asymmetric features that determine regulatory mechanisms and cellular functions in many signaling networks. The recent crystal structures of the mitochondrial Hsp90 isoform TRAP1 in complexes with ATP analogs have provided first evidence of significant asymmetry in the closed dimerized state that triggers independent activity of the chaperone protomers, whereby preferential hydrolysis of the buckled protomer is followed by conformational flipping between protomers and hydrolysis of the second protomer. Despite significant insights in structural characterizations of the TRAP1 chaperone, the atomistic details and mechanics of allosteric interactions that couple sequential ATP hydrolysis with asymmetric conformational switching in the TRAP1 protomers remain largely unknown. In this work, we explored atomistic and coarse-grained simulations of the TRAP1 dimer structures in combination with the ensemble-based network modeling and perturbation response scanning of residue interaction networks to probe salient features underlying allosteric signaling mechanism. This study has revealed that key effector sites that orchestrate allosteric interactions occupy the ATP binding region and N-terminal interface of the buckled protomer, whereas the main sensors of allosteric signals that drive functional conformational changes during ATPase cycle are consolidated near the client binding region of the straight protomer, channeling the energy of ATP hydrolysis for client remodeling. The community decomposition analysis of the interaction networks and reconstruction of allosteric communication pathways in the TRAP1 structures have quantified mechanism of allosteric regulation, revealing control points and interactions that coordinate asymmetric switching during ATP hydrolysis.

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production.

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.

ATPase Domain of Heat Shock protein 70—isoform 2—(Hsp70-2) and their role in activating the adaptive immune response: An in silico approach

A lot of molecules play fundamental roles in neoplasic processes and cancer. Heat shock proteins may enhance this severe pathology and favor the protumoral milieu. However, a look at the literature tells us that these molecules intervene in both to promote or attack cancer cells. In the case of breast cancer is known that Hsp70 (isoform 2) improve this establishment and progression in the patient, and is possible that the ATPase domain of Hsp70-2 favors this disease. Thus, is relevant to know if this molecular region has immunogenic activity as well as which epitopes are essential to stimulate immune cells, and whether could induce the attack of the tumor mass. In this aim, the immunogenicity of ATPase domain of Hsp70-2 was studied in silico . The results suggest that the majority of the molecule had immunogenic epitopes that boosts the immune response through activation of B cells and T cells. However, in vitro synthesis and in vivo experimental studies to evaluate the efficacy of this therapeutic candidate are required to ensure efficacy and safety in people.

Heat Shock Protein 90α, Assisted by Co‐Chaperones p23 and Cdc37, Promotes Opioid Anti‐Nociception in the Brain via Promoting ERK MAPK Signaling

Reducing opioid side effects like constipation and addiction has been a strong focus for improving opioid therapy in treating acute and chronic pain. One such approach includes manipulation of mu opioid receptor (MOR) signaling cascades, such as βarrestin2. However, the MOR signaling complex and mechanisms of signaling regulation are poorly understood. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator, which could be a target for improving the therapeutic profile of opioids. We found that intracerebroventricular (icv) 17‐AAG, a well‐established Hsp90 inhibitor, strongly blocked or abolished morphine anti‐nociception in paw incision and HIV neuropathic pain in mice. The current study was designed to identify molecular mechanisms of this regulation, including which isoform(s) and/or co‐chaperone(s) are involved in the regulatory effect of Hsp90 on opioid signaling. Male and female CD‐1 mice were treated with selective inhibitors for different Hsp90 isoforms (KU‐A, KUNB106, KUNG65) and co‐chaperones (KU177, gedunin, celastrol) via icv injection. We found that an Hsp90α selective inhibitor blocked morphine anti‐nociception by ~ 80% in the paw incision model, however, inhibitors for Hsp90β and Grp94 had no impact on morphine anti‐nociception. Knockdown of Hsp90α in the brain by CRISPR‐Cas9 also showed strong inhibition of morphine anti‐nociception in paw incision, confirming the results. Further, inhibition of the co‐chaperones p23 and Cdc37, but not Aha1, showed similar effects as Hsp90α inhibition in the paw incision model. To determine the molecular mechanism of these activities, we found that Hsp90α inhibitor treatment blocked ERK and JNK MAPK activation induced by the selective MOR agonist DAMGO without changing the expression of Hsp70 in mouse periaqueductal grey (PAG). Treatment with the ERK MAPK inhibitor U0126 recapitulated our results of Hsp90 inhibition on paw incision, HIV neuropathic, and tail flick pain, suggesting that ERK MAPK regulation is the means by which Hsp90α regulates morphine anti‐nociception. Identification of the isoforms and co‐chaperones involved in Hsp90 regulation of ERK and opioid anti‐nociception may permit more selective manipulation of the MOR signaling machinery to improve opioid efficacy and reduce side effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hsp90\u03b2 knockdown in DIO mice reverses insulin resistance and improves glucose tolerance

BackgroundInhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism.MethodsIn a diet-induced obese (DIO) mouse model of diabetes, expression of various Hsp90 isoforms in skeletal tissue was examined. Subsequent experiments characterized the role of Hsp90ab1 isoform in glucose metabolism and insulin signaling in primary human skeletal muscle myoblasts (HSMM) and a DIO mouse model.ResultsIn DIO mice Hsp90ab1 mRNA was upregulated in skeletal muscle compared to lean mice and knockdown using anti-sense oligonucleotide (ASO) resulted in reduced expression in skeletal muscle that was associated with improved glucose tolerance, reduced fed glucose and fed insulin levels compared to DIO mice that were treated with a negative control oligonucleotide. In addition, knockdown of HSP90ab1 in DIO mice was associated with reduced pyruvate dehydrogenase kinase-4 mRNA and phosphorylation of the muscle pyruvate dehydrogenase complex (at serine 232, 293 and 300), but increased phosphofructokinase 1, glycogen synthase 1 and long-chain specific acyl-CoA dehydrogenase mRNA. In HSMM, siRNA knockdown of Hsp90ab1 induced an increase in substrate metabolism, mitochondrial respiration capacity, and insulin sensitivity, providing further evidence for the role of Hsp90ab1 in metabolism.ConclusionsThe data support a novel role for Hsp90ab1 in arbitrating skeletal muscle plasticity via modulation of substrate utilization including glucose and fatty acids in normal and disease conditions. Hsp90ab1 represents a novel target for potential treatment of metabolic disease including diabetes.

Structure-guided design of an Hsp90\u03b2 N-terminal isoform-selective inhibitor

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone responsible for folding proteins that are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms. pan-Inhibition of Hsp90 appears to be detrimental as toxicities have been reported alongside induction of the pro-survival heat shock response. The development of Hsp90 isoform-selective inhibitors represents an alternative approach towards the treatment of cancer that may limit some of the detriments. Described herein is a structure-based approach to design isoform-selective inhibitors of Hsp90β, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90β-selective inhibitors as a method to overcome the detriments associated with pan-inhibition.

Interaction of the middle domains stabilizes Hsp90α dimer in a closed conformation with high affinity for p23.

The human genome encodes two highly similar cytosolic Hsp90 proteins called isoforms Hsp90α and Hsp90β. Of the 300 client proteins for Hsp90 identified so far only a handful interact specifically with one Hsp90 isoform. Here we report for the first time that Hsp90 cochaperone p23 binds preferentially to Hsp90α and that this interaction is mediated by the middle domain of Hsp90α. Based on the homology modeling, we infer that the middle domains in the Hsp90α dimer bind stronger with each other than in the Hsp90β dimer. Therefore, compared to Hsp90β, Hsp90α may adopt closed conformation more easily. Hsp90 interacts with p23 in the closed conformation. Hsp90α binds human recombinant p23 about three times stronger than Hsp90β but with significantly smaller exothermic enthalpy as determined by isothermal titration calorimetry of direct binding between the purified proteins. As p23 binds to Hsp90 in a closed conformation, stabilization of the Hsp90α dimer in the closed conformation by its middle domains explains preference of p23 to this Hsp90 isoform.

Could Targeting the Heat Shock Protein 90 Revolutionize Antiviral therapy?

Traditional antiviral strategies that target viral components are frequently associated with the generation of drug-resistant viruses. Thus, the development of novel antiviral drugs is critical. Hsp90 is a promising broad-spectrum antiviral drug target; however, whether targeting Hsp90 will revolutionize antiviral therapy remains ambiguous. Here, we summarize how Hsp90 functions in relation to its interactors, and listed the specific Hsp90 isoforms that participated in the virus life cycle. We also discuss the advantages and challenges of targeting Hsp90, taking into account antiviral activity, toxicity and the likelihood of emergence of drug-resistant viruses. Overall, we highlight that targeting Hsp90 might represent a novel and effective antiviral strategy. However, further studies are required before Hsp90 inhibitors can be used in antiviral therapy.

UCS protein function is partially restored in the Saccharomyces cerevisiae she4 mutant with expression of the human UNC45-GC, but not UNC45-SM

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90β isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90β. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90β in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved—yeast to man—in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking—events also facilitated by the myosins in yeast.

(-)-Epigallocatechin-3-Gallate Inhibits the Chaperone Activity of Plasmodium falciparum Hsp70 Chaperones and Abrogates Their Association with Functional Partners.

Heat shock proteins (Hsps), amongst them, Hsp70 and Hsp90 families, serve mainly as facilitators of protein folding (molecular chaperones) of the cell. The Hsp70 family of proteins represents one of the most important molecular chaperones in the cell. Plasmodium falciparum, the main agent of malaria, expresses six Hsp70 isoforms. Two (PfHsp70-1 and PfHsp70-z) of these localize to the parasite cytosol. PHsp70-1 is known to occur in a functional complex with another chaperone, PfHsp90 via a co-chaperone, P. falciparum Hsp70-Hsp90 organising protein (PfHop). (−)-Epigallocatechin-3-gallate (EGCG) is a green tea constituent that is thought to possess antiplasmodial activity. However, the mechanism by which EGCG exhibits antiplasmodial activity is not fully understood. A previous study proposed that EGCG binds to the N-terminal ATPase domain of Hsp70. In the current study, we overexpressed and purified recombinant forms of two P. falciparum cytosol localized Hsp70s (PfHsp70-1 and PfHsp70-z), and PfHop, a co-chaperone of PfHsp70-1. Using the surface plasmon resonance approach, we demonstrated that EGCG directly binds to the two Hsp70s. We further observed that binding of EGCG to the two proteins resulted in secondary and tertiary conformational changes. In addition, EGCG inhibited the ATPase and chaperone function of the two proteins. Furthermore, EGCG abrogated association of the two Hsp70s with their functional partners. Using parasites cultured in vitro at the blood stages, we observed that 2.9 µM EGCG suppressed 50% P. falciparum parasite growth (IC50). Our findings demonstrate that EGCG directly binds to PfHsp70-1 and PfHsp70-z to inhibit both the ATPase and chaperone functions of the proteins. Our study constitutes the first direct evidence suggesting that the antiplasmodial activity of EGCG is at least in part accounted for by its inhibition of Hsp70 function.

Role of intestinal Hsp70 in barrier maintenance: contribution of milk to the induction of Hsp70.2.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease of complex etiology resulting in devastating systemic inflammation and often death in premature newborns. We previously demonstrated that formula feeding inhibits ileal expression of heat shock protein-70 (Hsp70), a critical stress protein within the intestine. Barrier function for the premature intestine is critical. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability. Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function as well as intestine from Hsp70-/- pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70. Immunostaining demonstrates co-localized Hsp70 and tight junction protein zona occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM. RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3, predominate in WT pup; however, Hsp70.2 predominates in the KO pups. While Hsp70 is present in WT milk, it is not present in KO EM. Hsp70 associates with ZO-1 to maintain epithelial barrier function. Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine, suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother's milk. These results suggest that mother's milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC. Level III.

Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.