scholarly journals Twin-arginine translocase component TatB performs folding quality control via a general chaperone activity

2020 ◽  
Author(s):  
May N. Taw ◽  
Jason T. Boock ◽  
Daniel Kim ◽  
Mark A. Rocco ◽  
Dujduan Waraho-Zhmayev ◽  
...  
Keyword(s):  
Quality Control ◽  
Citrate Synthase ◽  
Active Species ◽  
Chaperone Activity ◽  
Twin Arginine Translocation ◽  
Substrate Protein ◽  
Twin Arginine Translocase ◽  
Partially Unfolded

AbstractThe twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details of this QC mechanism remain poorly understood. Here, we hypothesized that the conformational state of Tat substrates is directly sensed by the TatB component of the bacterial Tat translocase. In support of this hypothesis, several TatB variants in which the cytoplasmic membrane-extrinsic domain was either truncated or mutated in the vicinity of a conserved, highly flexible α-helical domain were observed to form functional translocases in vivo that had compromised QC activity as evidenced by the uncharacteristic export of several misfolded protein substrates. In vitro folding experiments revealed that the membrane-extrinsic domain of TatB possessed general chaperone activity, transiently binding to highly structured, partially unfolded intermediates of a model protein, citrate synthase, thereby preventing its irreversible aggregation and stabilizing the active species. Collectively, these results suggest that the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates.

scholarly journals Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox

2003 ◽  
Vol 374 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Miki OKADA ◽  
Hideaki ITOH ◽  
Takashi HATAKEYAMA ◽  
Hiroshi TOKUMITSU ◽  
Ryoji KOBAYASHI
Keyword(s):  
Citrate Synthase ◽  
Heat Shock Protein 90 ◽  
Chaperone Activity ◽  
Physiological Significance ◽  
Disodium Cromoglycate ◽  
Terminal Domain ◽  
C Terminus

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5–3.0 μM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

scholarly journals DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone

2004 ◽  
Vol 383 (1) ◽  
pp. 165-170 ◽  
Author(s):  
Attila FARKAS ◽  
Gábor NARDAI ◽  
Peter CSERMELY ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Heat Shock ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Citrate Synthase ◽  
Sequence Similarity ◽  
Heat Shock Protein 90 ◽  
Chaperone Activity ◽  
In Vitro Assays

UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

scholarly journals Humanin is an endogenous activator of chaperone-mediated autophagy

2017 ◽  
Vol 217 (2) ◽  
pp. 635-647 ◽  
Author(s):  
Zhenwei Gong ◽  
Inmaculada Tasset ◽  
Antonio Diaz ◽  
Jaime Anguiano ◽  
Emir Tas ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Death ◽  
Heat Shock Protein 90 ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Selective Degradation ◽  
Cytosolic Side ◽  
Chaperone Mediated Autophagy

Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects.

Abstract 420: Delineating the Caspase-dependent Targets and Signal Pathways That Promote Pathological Cardiac Hypertrophy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Charis Putinski ◽  
Mohammad Abdul-Ghani ◽  
Rebecca Stiles ◽  
Steve Brunette ◽  
Sarah A Dick ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Pathological Process ◽  
Signal Pathways ◽  
Caspase Cleavage ◽  
Substrate Protein ◽  
Cell Death Pathway ◽  
Effector Caspase ◽  
The Impact

Although cardiac hypertrophy is initially an adaptive response, chronic stress on the heart is a maladaptive process that inevitably leads to end-stage heart failure. Interestingly, this pathological process is also characterized by cell behaviors associated with apoptosis. We previously demonstrated the essential role of the intrinsic cell death pathway during cardiac hypertrophy; however, the caspase-dependent pathways and cleavage targets remain elusive. To this aim, we evaluated a myocyte enhancer factor 2 (MEF2) transcription factor inhibitor, histone deacetylase 3 (HDAC3), and gelsolin as potential caspase cleavage substrates involved in the induction and/or maintenance of cardiac hypertrophy. In vitro cleavage assays were completed with effector caspase and recombinant substrate protein which demonstrated caspase-dependent cleavage. HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Luciferase assays demonstrated that the transcriptional activity of MEF2 is dependent on intact caspase function suggesting caspase-directed HDAC3 cleavage may serve as a novel regulatory mechanism to alleviate MEF2 suppression to engage the hypertrophy gene expression program. Unlike HDAC3, caspase mediated gelsolin cleavage occurs at latter stages and is coincident with the cytoskeletal alterations that occur during this process. As gelsolin is a potent actin capping/severing enzyme, we hypothesize that caspase-mediated gelsolin activation acts as a key regulatory step in the structural rearrangements that allow for hypertrophy to occur. We have generated adenoviral vectors containing caspase cleavage mutants and cleaved forms of HDAC3 and gelsolin and will discuss the impact of these modified substrates on the hypertrophy process in vitro and in vivo. Collectively, this work suggests that caspase signalling acts to engage both the transcriptional program and cytoskeletal accommodations that characterize cardiac hypertrophy. Importantly, these observations suggest that identification of inhibitors that suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.

Preparation of fibrils for intracerebral injection v1

2021 ◽  
Author(s):  
The Michael J Fox Foundation Pff Standardization Consortium
Keyword(s):  
Quality Control ◽  
Best Practices ◽  
External Validation ◽  
Alpha Synuclein ◽  
Intracerebral Injection ◽  
Primary Neuron ◽  
Consensus Protocol ◽  
Reference Section

This is a consensus protocol developed through discussions with Laura Volpicelli-Daley, Caryl Sortwell, Kelvin Luk, Lindsey Gottler, and Virginia Lee. This protocol is intended for research purposes only, using specially-formulated monomeric alpha-synuclein protein available for purchase at Proteos, Inc as the result of efforts by The Michael J. Fox Foundation (MJFF). Each batch of the “Alpha-Synuclein Monomer Protein for Making Pre- Formed Fibrils” has undergone internal purification and quality control at Proteos in addition to external validation to confirm successful generation of pathogenic aSyn PFFs. See Reference section for methods and results from application of alpha-synuclein pre-formed fibrils (aSyn PFFs) in primary neuron cultures in vitro or in mice in vivo. This protocol is referenced in the Polinski et al 2018 paper entitled "Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents" (doi: 10.3233/JPD-171248).

Protocol for Generation of Pre-Formed Fibrils from Alpha-Synuclein Monomer v1

2021 ◽  
Author(s):  
The Michael J Fox Foundation Pff Standardization Consortium
Keyword(s):  
Parkinson’S Disease ◽  
Quality Control ◽  
Best Practices ◽  
External Validation ◽  
Alpha Synuclein ◽  
Primary Neuron ◽  
Consensus Protocol ◽  
Reference Section

This is a consensus protocol developed through discussions with Laura Volpicelli-Daley, Caryl Sortwell, Kelvin Luk, Lindsey Gottler, and Virginia Lee. This protocol is intended for research purposes only, using specially-formulated monomeric alpha-synuclein protein available for purchase at Proteos, Inc as the result of efforts by The Michael J. Fox Foundation (MJFF). Each batch of the “Alpha-Synuclein Monomer Protein for Making Pre- Formed Fibrils” has undergone internal purification and quality control at Proteos in addition to external validation to confirm successful generation of pathogenic aSyn PFFs. See Reference section for methods and results from application of alpha-synuclein pre-formed fibrils (aSyn PFFs) in primary neuron cultures in vitro or in mice in vivo. This protocol is referenced in the Polinski et al 2018 paper entitled "Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents" (doi: 10.3233/JPD-171248).

Marsdenia tenacissima: A Review of Traditional Uses, Phytochemistry and Pharmacology

2018 ◽  
Vol 46 (07) ◽  
pp. 1449-1480 ◽  
Author(s):  
Peile Wang ◽  
Jing Yang ◽  
Zhenfeng Zhu ◽  
Xiaojian Zhang
Keyword(s):  
Quality Control ◽  
Multidrug Resistance ◽  
Control Method ◽  
Food Poisoning ◽  
Bioactive Constituents ◽  
Resistance Reversal ◽  
Wide Clinical Application ◽  
Marsdenia Tenacissima

The stems and roots of Marsdenia tenacissima (Roxb.) Wight et Arn., a traditional Chinese medicine and Dai herbal medicine, have been widely used for the treatment of asthma, trachitis, tonsillitis, pharyngitis, cystitis, pneumonia and drug or food poisoning. Nowadays, the extract of Marsdenia tenacissima, under the trademark of “Xiao-ai-ping”, is widely used in clinic for the treatment of different cancers in China. To date, approximately 196 chemical ingredients covering steroids, triterpenes and organic acids have been identified from different parts of this plant. Steroids are the major characteristic and bioactive constituents of this plant. Modern pharmacology has demonstrated that the crude extracts and steroids have various in vitro and in vivo pharmacological activities, such as multidrug resistance reversal, antitumor, anti-angiogenic, immunomodulation and anti-HIV activities. The multidrug resistance reversal of steroids provided evidence for the use of this herb in clinic. However, despite wide clinical application, clinical trials, quality control method, pharmacokinetic and toxicity research on Marsdenia tenacissima were seldom reported and deserved further efforts. The present review aimed to achieve a comprehensive and up-to-date investigation in ethnopharmacology, phytochemistry, pharmacology, clinical study, pharmacokinetics, toxicology and quality control of Marsdenia tenacissima. In addition, the possible perspectives and trends for future studies of Marsdenia tenacissima have also been put forward. It is believed that this review would provide a theoretical basis and valuable data for future in-depth studies and applications.

Chemical assays--new opportunities.

1987 ◽  
Vol 33 (9) ◽  
pp. 1574-1578 ◽  
Author(s):  
A E Burkhardt
Keyword(s):  
Quality Control ◽  
Solid Phase ◽  
Clinical Chemistry ◽  
Test Format ◽  
Home Testing

Abstract The acceptance of the solid-phase format in various areas of clinical chemistry is the consequence of the advantages of this test format, which include stability of the reagents, unitized packaging, convenient and small instruments, and minimal preparations by users before testing. Overall, these advantages provide very convenient tests. Future successful uses of solid-phase reagents depend upon how well these features meet the needs of the users. Needs for systems to be used in the decentralized laboratory include even less cost, even more convenience, and improved quality control. Needs for home testing include convenient tests with clinically useful accuracy, improved quality control, and improved recording systems to overcome user bias in recording results. New solid-phase technologies being developed include noncolorimetric systems suitable for use with miniature probes, for in vitro or in vivo use, and spectrophotometric systems for determinations of analytes directly in capillaries of the skin without invasive sampling.

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