The Sigma-1 Receptor as a Pharmacologic Chaperone: Energetics

Initially thought to be an opioid receptor subtype, Sigma-1 receptors (S1R) are now known to be unique proteins that have chaperone-like properties. As such, they play critical roles in cellular signaling, homeostasis, and cell survival. These roles offer significant insight for understanding homeostasis of normal physiologic processes, and the pathophysiologic consequences of disruption of normal function. Because of the broad nature of chaperone action, S1R agonists and antagonists represent potential drug discovery goals for the pharmacotherapeutic treatment of a variety of disorders that result from dysfunctional proteins. The present study summarizes the S1R as a pharmacologic chaperone crucial for protein folding and cellular homeostasis. Through literature review and thermodynamic analysis, it explores how S1R stabilizes target proteins, influencing neuroprotection and potential drug therapies. The binding of chaperones to target proteins is thermodynamically favorable, offering insights into treating diseases linked to protein misfolding.

Molecular chaperones in stroke-induced immunosuppression

Stroke-induced immunosuppression is a process that leads to peripheral suppression of the immune system after a stroke and belongs to the central nervous system injury-induced immunosuppressive syndrome.Stroke-induced immunosuppression leads to increased susceptibility to post-stroke infections,such as urinary tract infections and stroke-associated pneumonia,worsening prognosis.Molecular chaperones are a large class of proteins that are able to maintain proteostasis by directing the folding of nascent polypeptide chains,refolding misfolded proteins,and targeting misfolded proteins for degradation.Various molecular chaperones have been shown to play roles in stroke-induced immunosuppression by modulating the activity of other molecular chaperones,cochaperones,and their associated pathways.This review summarizes the role of molecular chaperones in stroke-induced immunosuppression and discusses new approaches to restore host immune defense after stroke.

Effect of chaperone–client interaction strength on Hsp70-mediated protein folding

Protein folding in crowding cellular environment often relies on the assistance of various chaperones. Hsp70 is one of the most ubiquitous chaperones in cells. Previous studies showed that the chaperone–client interactions at the open state tend to remodel the protein folding energy landscape and direct the protein folding as a foldase. In this work, we further investigate how the chaperone–client interaction strength modulates the foldase function of Hsp70 by using molecular simulations. The results showed that the time of substrate folding(including the whole folding step and substrate release step) has a non-monotonic dependence on the interaction strength. With the increasing of the chaperone–client interaction strength, the folding time decreases first, and then increases. More detailed analysis showed that when the chaperone–client interaction is too strong, even small number of chaperones–client contacts can maintain the substrate bound with the chaperone. The sampling of the transient chaperones–client complex with sparse inter-molecule contacts makes the client protein have chance to access the misfolded state even it is bound with chaperone. The current results suggest that the interaction strength is an important factor controlling the Hsp70 chaperoning function.

Roles of constitutively secreted extracellular chaperones in neuronal cell repair and regeneration

Protein quality control involves many processes that jointly act to regulate the expression, localization, turnover, and degradation of proteins, and has been highlighted in recent studies as critical to the differentiation of stem cells during regeneration. The roles of constitutively secreted extracellular chaperones in neuronal injury and disease are poorly understood. Extracellular chaperones are multifunctional proteins expressed by many cell types, including those of the nervous system, known to facilitate protein quality control processes. These molecules exert pleiotropic effects and have been implicated as playing important protective roles in a variety of stress conditions, including tissue damage, infections, and local tissue inflammation. This article aims to provide a critical review of what is currently known about the functions of extracellular chaperones in neuronal repair and regeneration and highlight future directions for this important research area. We review what is known of four constitutively secreted extracellular chaperones directly implicated in processes of neuronal damage and repair, including transthyretin, clusterin, α2-macroglobulin, and neuroserpin, and propose that investigation into the effects of these and other extracellular chaperones on neuronal repair and regeneration has the potential to yield valuable new therapies.

Ischemic preconditioning induces chaperone hsp70 expression and inhibits protein aggregation in the CA1 neurons of rats

Objective To investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism. Methods Two-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10- min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions. Results Histological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region （P 〈 0.01 vs 10-min ischemia group）. Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates（P〈0.01 vs 10-min ischemia group）.Conelusion Ischemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

AtFKBP53 is a histone chaperone required for repression of ribosomal RNA gene expression in Arabidopsis

Chromatin structure is important for controlling gene expression, but mechanisms underlying chromatin remodel- ing are not fully understood. Here we report that an FKBP （FK506 binding protein） type immunophilin, AtFKBP53, possesses histone chaperone activity and is required for repressing ribosomal gene expression in Arabidopsis. The At- FKBP53 protein is a multidomain FKBP with a typical peptidylprolyl isomerase （PPIase） domain and several highly charged domains. Using nucleosome assembly assays, we showed that AtFKBP53 has histone chaperone activity and the charged acidic domains are sufficient for the activity. We show that AtFKBP53 interacts with histone H3 through the acidic domains, whereas the PPIase domain is dispensable for histone chaperone activity or histone binding. Ri- bosomal RNA gene （18S rDNA） is overexpressed when AtFKBP53 activity is reduced or eliminated in Arabidopsis plants. Chromatin immunoprecipitation assay showed that AtFKBP53 is associated with the 18S rDNA gene chro- matin, implicating that AtFKBP53 represses rRNA genes at the chromatin level. This study identifies a new histone chaperone in plants that functions in chromatin remodeling and regulation of transcription.

Chaperones in hepatitis C virus infection

The hepatitis C virus(HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases:(1) binding and internalization;(2) cytoplasmic release and uncoating;(3) viral polyprotein translation and processing;(4) RNA genome replication;(5) encapsidation(packaging) and assembly; and(6) virus morphogenesis(maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Molecular chaperones and hypoxic-ischemic encephalopathy

Hypoxic-ischemic encephalopathy（HIE） is a disease that occurs when the brain is subjected to hypoxia,resulting in neuronal death and neurological deficits,with a poor prognosis.The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release,cellular proteolysis,reactive oxygen species generation,nitric oxide synthesis,and inflammation.The molecular and cellular changes in HIE include protein misfolding,aggregation,and destruction of organelles.The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway,the extrinsic Fas receptor pathway,and the endoplasmic reticulum stress-induced pathway.Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century.Hypothermia,xenon gas treatment,the use of melatonin and erythropoietin,and hypoxic-ischemic preconditioning have proven effective in HIE patients.Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes.A large number of molecular chaperones are induced after brain ischemia and hypoxia,among which the heat shock proteins are the most important.Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects.Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations,assisting in the proper folding of newly synthesized polypeptides,regulating the degradation of misfolded proteins,inhibiting the aggregation of proteins,and by controlling the refolding of misfolded proteins.In addition,heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways,including the intrinsic pathway,the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway.Molecular chaperones play a key role in neuroprotection in HIE.In this review,we provide an overview of the mechanisms of HIE and discuss the various treatment strategies.Given their critical role in the disease,molecular chaperones are promising therapeutic targets for HIE.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Orf1/SpcS Chaperones ExoS for Type Three Secretion by Pseudomonas aeruginosa

Objective Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type Ⅲ secretion system （TTSS） to inject effector proteins directly into the cytosol of target cells to subvert the host cell＇s functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P aeruginosa, we analyzed the role of a postulated chaperone termed Orfl. Methods By allelic exchange, we constructed the mutant with the deletion of gene Orfl. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orfl, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF The role of Orfl in the expression of exoS was evaluated by gene reporter analysis. Results Pull-down assay showed that Orfl binds to ExoS and ExoT. Secretion profile analysis showed that Orfl was necessary for the optimal secretion of ExoS and ExoT. However, Orfl had no effect on the expression of exoS. Conclusion Orfl is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orfl as SpcS for ＂specific Pseudomonas chaperone for ExoS＂.

Molecular chaperones and neurodegenerative diseases

Neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates that result from conformational changes in proteins. These diseases may result from an imbalance between the produetion of misfolded proteins and normal chaperone capacity. Molecular chaperones provide a first line of defenee against misfolded, aggragation-prone proteins and are, therefore, promising therapeutic targets for neurodegenerative diseases.

Single-step Purification of Molecular Chaperone GroEL by Expanded Bed Chromatography

Expanded bed adsorption (EBA) is an integrative downstream processing technique for the purification of biological substances directly from unclarified feedstock. In this study, molecular chaperone GroEL, an important protein folding helper both in vivo and in vitro, was purified by the single-step EBA technique from the unclarified homogenate of recombinant E. coli cells. Compared with packed bed adsorption, the EBA technique provided a single-step approach to yield an electrophoretic purity of GroEL. After the homogenate loading and column washing in the expanded bed mode, the GroEL protein was recovered by stepwise salt-gradient elution in packed-bed or expanded-bed modes, respectively. The expanded-bed elution mode was found as efficient as the packed-bed mode in the purification of GroEL from cell disruptate.

Oxidative modification of the molecular chaperone family in a PC12 cell model of Parkinson's disease induced by Z-Ile-Glu(OtBu)-Ala-Leucinal

Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson＇s disease patients. In the present study, proteasome inhibitor Z-Ile-Glu（OtBu）-Ala-Leucinal （PSI） was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson＇s disease. Oxidatively modified proteins were identified to determine pathogenesis of Parkinson＇s disease. Results demonstrated that 24 hours of 10 IJmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson＇s disease： neuronal degeneration and eosinophilic inclusion formation in neurons. In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected： chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58, and heat shock protein 70. This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson＇s disease induced with PSI.

Differential Expression of Molecular Chaperones in PC12 Cells Treated with PSI

Parkinson＇s disease（PD） is a common neurodegenerative disorder whose primary pathology features are the degeneration of dopaminergic neurons in the substantia nigra pars compacta（SNc） and the presence of eosinophilic inclusions called Lewy body in the cytoplasm of the remained neurons. Growing evidence suggests that dysfunction of the ubiquitin-proteasome system（UPS） is involved in the etiopathogenesis of PD. In order to investigate the pathogenetic mechanism of ubiquitin-proteasome dysfunction in PD, 2D-differential gel electrophoresis（2D-DIGE） and MALDI-TOF Pro MS were used to determine the proteins, which were differentially expressed, in PC12 cells that had undergone a synthetic proteasomal inhibitor PSI（10 μmol/L） treatment for 24 h. Forty-six protein spots were differentially expressed in response to PSI administration, of which 34 were increased and 12 decreased. Six of these were identified as molecular charperones： endoplasmin precursor（GRP94）, heat shock protein 105（HSP105）, HSC-70-psl, glucose ruglated protein 75（GRP75）, glucose ruglated protein 58（GRP58） and heat shock 27000 protein l（HSP27）. The results suggest that the molecular chaperones play an important role in the PD model induced by proteasomal inhibitor.

GroESL protects superoxide dismutase (SOD)— Deficient cells against oxidative stress and is a chaperone for SOD

Superoxide dismutase (SOD)-deficient Escherichia coli OX326Acells are protected against chemically-induced oxidative stress by expression of the chaperonin GroESL. This protection is equivalent to expression of superoxide dismutase even though GroESL has no inherent SOD activity. Co-overexpression of GroESL and SOD in the same cells results in higher protein yields of SOD and greater metallation of SOD when compared with expression of SOD alone. Greater metallation results in the higher specific activity of SOD that is observed in heat shock, and is not due to increased synthesis of SOD mRNA or protein.

CD8 T cell response in a phase I study of therapeutic vaccination of advanced NSCLC with allogeneic tumor cells secreting endoplasmic reticulum-chaperone gp96-Ig-peptide complexes

Antigen containing, allogeneic cells secreting the genetically modified protein and peptide-chaperone gp96-Ig cross, prime and expand antigen specific CD8 T cells with therapeutic antitumor activity in mice. In a first in man phase I study, we now report the results of therapeutic vaccination of non-small cell lung cancer (NSCLC) patients with an established, allogeneic non-small cell lung adenocarcinoma cell line secreting gp96-Ig. Advanced NSCLC-patients stage IIIB or IV of any histological subtype were enrolled and treated with up to 36 vaccinations over the course of 18 weeks. Primary endpoint was safety, secondary endpoints tumor response and overall survival. Measurement of tumor antigen specific CD8 CTL responses is precluded by the lack of known NSCLC associated antigens. Therefore, we measured patient CD8 T cell-IFN-γ responses to allo-antigens of the vaccine cells as surrogate for tumor antigen specific CD8 CTL. In 7 of 18 treated patients tumor growth was stabilized, however none of the 18 patients had an objective tumor response by RECIST criteria. Of 15 patients evaluable for immune response, 11 responded to vaccination with more than twofold increase in CD8-IFN-γ frequency above baseline. These patients had a median survival time of 16.5 months. Four patients who had no CD8 response above base line had survival times from 2.1 to 6.7 months. Our data are consistent with the concept that generation of CD8 CTL by therapeutic vaccination may delay tumor growth and progression and mediate prolonged survival even in the absence of objective tumor responses. Further studies will be required to test this concept and promising result.

A Role for Histone Chaperones in Regulating RNA Polymerase II

Transcription is a highly regulated cellular process in which dysfunction leads to disease. One level of regulation is chromatin structure which protects promoters from transcription factor binding. To circumvent this blockade, histone chaperones aid in displacement of nucleosomes. In particular, the histone chaperone complex HUCA, consisting of Hira, Ubn1, Cabin1, and ASF1a, replaces histone variant H3.1 with H3.3 in front of actively transcribing RNA Polymerase II (RNAPII). The 26S proteasome is a major degrader of proteins within the cell and plays both proteolytic and non- proteolytic roles in transcriptional regulation. One major role is the degradation of irreversibly arrested RNAPII. Several interactions between HUCA, the 26S proteasome, and RNAPII have been characterized individually;we now present observations from our lab and others which directly associate elongating RNAPII with the degradation machinery through observations of involvement with the HUCA complex. Our short report presents these ideas and discusses their importance in transcriptional regulation as well as implications in disease manifestation.

AAA+ ClpB chaperone as a potential virulence factor of pathogenic microorganisms: Other aspect of its chaperone function

We describe and discuss the most recent findings on the activity and function of the oligomeric AAA+ chaperone ClpB from the Hsp100 protein family in pathogenic microorganisms. Pathogens are exposed to significant stress during infection of the host cells, frequently resulting in protein aggregation. The fact that ClpB is usually up-regulated in pathogens together with its immune reactivity suggests that ClpB acting as a protein disaggregase may be important for pathogen invasion and virulence. However, the specific function of ClpB in pathogenicity is still unclear. Since it is known that ClpB does not exist in mammals, it may serve as a potential target for the development of an effective therapy against several major bacterial diseases that do not respond to conventional antibiotics.