-
Journal of Visualized Experiments : JoVE Jan 2012This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with...
This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding and have a crucial role in promoting cell survival upon stress insults like heat shock, nutrient starvation and exposure to chemicals/poisons. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells as well as neurodegeneration. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy and neurodegenerative disorders. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).
Topics: Enzymes; HEK293 Cells; Heat-Shock Response; Humans; Luciferases, Firefly; Luciferases, Renilla; Molecular Chaperones; Protein Refolding; Transfection
PubMed: 22297799
DOI: 10.3791/3540 -
Medicinal Research Reviews Nov 2022The 94 kDa molecular chaperone, glucose-regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic... (Review)
Review
The 94 kDa molecular chaperone, glucose-regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic reticulum (ER) stress and disease. Grp94 belongs to the Hsp90 family of molecular chaperones and is a master regulator of ER homeostasis due to its ability to fold and stabilize proteins/receptors, and to chaperone misfolded proteins for degradation. Multiple studies have demonstrated that Grp94 knockdown or inhibition leads to the degradation of client protein substrates, which leads to disruption of disease-dependent signaling pathways. As a result, small molecule inhibitors of Grp94 have become a promising therapeutic approach to target a variety of disease states. Specifically, Grp94 has proven to be a promising target for cancer, glaucoma, immune-mediated inflammation, and viral infection. Moreover, Grp94-peptide complexes have been utilized effectively as adjuvants for vaccines against a variety of disease states. This work highlights the significance of Grp94 biology and the development of therapeutics that target this molecular chaperone in multiple disease states.
Topics: Biology; HSP70 Heat-Shock Proteins; Humans; Membrane Glycoproteins; Membrane Proteins; Molecular Chaperones
PubMed: 35861260
DOI: 10.1002/med.21915 -
The Journal of Biological Chemistry Feb 2023An extracellular network of molecular chaperones protects a diverse array of proteins that reside in or pass through extracellular spaces. Proteins in the extracellular... (Review)
Review
An extracellular network of molecular chaperones protects a diverse array of proteins that reside in or pass through extracellular spaces. Proteins in the extracellular milieu face numerous challenges that can lead to protein misfolding and aggregation. As a checkpoint for proteins that move between cells, extracellular chaperone networks are of growing clinical relevance. J-domain proteins (JDPs) are ubiquitous molecular chaperones that are known for their essential roles in a wide array of fundamental cellular processes through their regulation of heat shock protein 70s. As the largest molecular chaperone family, JDPs have long been recognized for their diverse functions within cells. Some JDPs are elegantly selective for their "client proteins," some do not discriminate among substrates and others act cooperatively on the same target. The realization that JDPs are exported through both classical and unconventional secretory pathways has fueled investigation into the roles that JDPs play in protein quality control and intercellular communication. The proposed functions of exported JDPs are diverse. Studies suggest that export of DnaJB11 enhances extracellular proteostasis, that intercellular movement of DnaJB1 or DnaJB6 enhances the proteostasis capacity in recipient cells, whereas the import of DnaJB8 increases resistance to chemotherapy in recipient cancer cells. In addition, the export of DnaJC5 and concurrent DnaJC5-dependent ejection of dysfunctional and aggregation-prone proteins are implicated in the prevention of neurodegeneration. This review provides a brief overview of the current understanding of the extracellular chaperone networks and outlines the first wave of studies describing the cellular export of JDPs.
Topics: Humans; Molecular Chaperones; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Proteostasis; Nerve Tissue Proteins
PubMed: 36581212
DOI: 10.1016/j.jbc.2022.102840 -
Biomolecules Nov 2022The core-1 β1-3galactosyltransferase-specific chaperone 1 (Cosmc) is a unique molecular chaperone of core-1 β1-3galactosyltransferase(C1GALT1), which typically... (Review)
Review
The core-1 β1-3galactosyltransferase-specific chaperone 1 (Cosmc) is a unique molecular chaperone of core-1 β1-3galactosyltransferase(C1GALT1), which typically functions inside the endoplasmic reticulum (ER). Cosmc helps C1GALT1 to fold correctly and maintain activity. It also participates in the synthesis of the T antigen, O-glycan, together with C1GALT1. Cosmc is a multifaceted molecule with a wide range of roles and functions. It involves platelet production and the regulation of immune cell function. Besides that, the loss of function of Cosmc also facilitates the development of several diseases, such as inflammation diseases, immune-mediated diseases, and cancer. It suggests that Cosmc is a critical control point in diseases and that it should be regarded as a potential target for oncotherapy. It is essential to fully comprehend Cosmc's roles, as they may provide critical information about its involvement in disease development and pathogenesis. In this review, we summarize the recent progress in understanding the role of Cosmc in normal development and diseases.
Topics: Humans; Endoplasmic Reticulum; Molecular Chaperones; Disease; Glycosylation
PubMed: 36551160
DOI: 10.3390/biom12121732 -
Journal of Molecular Biology Sep 2018The molecular chaperone Hsp90 is involved in the folding, maturation, and degradation of a large number structurally and sequentially unrelated clients, often connected... (Review)
Review
The molecular chaperone Hsp90 is involved in the folding, maturation, and degradation of a large number structurally and sequentially unrelated clients, often connected to serious diseases. Elucidating the principles of how Hsp90 recognizes this large variety of substrates is essential for comprehending the mechanism of this chaperone machinery, as well as it is a prerequisite for the design of client specific drugs targeting Hsp90. Here, we discuss the recent progress in understanding the substrate recognition principles of Hsp90 and its implications for the role of Hsp90 in the lifecycle of proteins. Hsp90 acts downstream of the chaperone Hsp70, which exposes its substrate to a short and highly hydrophobic cleft. The subsequently acting Hsp90 has an extended client-binding interface that enables a large number of low-affinity contacts. Structural studies show interaction modes of Hsp90 with the intrinsically disordered Alzheimer's disease-causing protein Tau, the kinase Cdk4 in a partially unfolded state and the folded ligand-binding domain of a steroid receptor. Comparing the features shared by these different proteins provides a picture of the substrate-binding principles of Hsp90.
Topics: Animals; Binding Sites; Carrier Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Hydrophobic and Hydrophilic Interactions; Molecular Chaperones; Protein Binding; Protein Interaction Domains and Motifs; Structure-Activity Relationship
PubMed: 29782836
DOI: 10.1016/j.jmb.2018.05.026 -
Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.Protein Science : a Publication of the... Jul 2024Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone...
Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
Topics: Molecular Chaperones; Binding Sites; Humans; Protein Domains; Adaptor Proteins, Signal Transducing; Models, Molecular; Hydrophobic and Hydrophilic Interactions
PubMed: 38864729
DOI: 10.1002/pro.5063 -
Biochemical Pharmacology Jul 2022The molecular chaperone protein HSP60 is mainly distributed in mitochondria and assists protein folding under physiological and pathological conditions. Accumulating... (Review)
Review
The molecular chaperone protein HSP60 is mainly distributed in mitochondria and assists protein folding under physiological and pathological conditions. Accumulating evidence suggests abnormally expressed HSP60 in cancer is associated with clinicopathological features and prognosis of cancer patients. HSP60 could be used as a new biomarker for both diagnostic and prognostic purpose and tumor therapy. In this review article, we briefly described the structure, functional cycle, and regulatory mechanism of HSP60, and summarized its functional diversity in cancer as well as recent progress related to the diagnostic application of HSP60 and inhibitors against HSP60, which could provide us a comprehensive understanding about the value of HSP60 in tumor management.
Topics: Chaperonin 60; Humans; Mitochondria; Molecular Chaperones; Neoplasms; Protein Folding
PubMed: 35609646
DOI: 10.1016/j.bcp.2022.115096 -
Cell Chemical Biology May 2022The molecular chaperone DnaK, is an attractive drug target for treating mycobacterial infections. In this issue, Hosfelt, Richards, and colleagues applied a...
The molecular chaperone DnaK, is an attractive drug target for treating mycobacterial infections. In this issue, Hosfelt, Richards, and colleagues applied a high-throughput screen and discovered inhibitors that disrupt cofactor-mediated activation of DnaK. These inhibitors can lower bacterial survival under stress and decrease resistance to key antibiotics.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Escherichia coli Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones
PubMed: 35594848
DOI: 10.1016/j.chembiol.2022.05.002 -
Biopolymers Aug 2016The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear... (Review)
Review
The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016.
Topics: Adenosine Triphosphatases; Animals; HSP90 Heat-Shock Proteins; Humans; Protein Conformation; Proteolysis
PubMed: 26991466
DOI: 10.1002/bip.22835 -
Philosophical Transactions of the Royal... May 2013The historical origins and current interpretation of the molecular chaperone concept are presented, with the emphasis on the distinction between folding chaperones and... (Review)
Review
The historical origins and current interpretation of the molecular chaperone concept are presented, with the emphasis on the distinction between folding chaperones and assembly chaperones. Definitions of some basic terms in this field are offered and misconceptions pointed out. Two examples of assembly chaperone are discussed in more detail: the role of numerous histone chaperones in fundamental nuclear processes and the co-operation of assembly chaperones with folding chaperones in the production of the world's most important enzyme.
Topics: Gene Expression Regulation; Histones; Molecular Chaperones; Protein Biosynthesis; Protein Conformation; Protein Folding
PubMed: 23530255
DOI: 10.1098/rstb.2011.0398