Analysis of Chaperone Function in Vitro

2008 ◽  
pp. 162-196 ◽  
Author(s):  
Johannes Buchner ◽  
Stefan Walter
Keyword(s):  
Chaperone Function

scholarly journals Effects of modifications of α-crystallin on its chaperone and other properties

2002 ◽  
Vol 364 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Barry K. DERHAM ◽  
John J. HARDING
Keyword(s):  
Congenital Cataract ◽  
Point Mutations ◽  
Small Heat Shock Protein ◽  
High Molecular Mass ◽  
Lens Crystallins ◽  
Post Translational Modifications ◽  
Chaperone Function ◽  
Arginine Residues

The role of α-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. α-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by ‘trapping’ the protein in a high-molecular-mass complex. However, during aging, the chaperone function of α-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts. Within the central part of the lens there is no turnover of damaged protein, and therefore post-translational modifications of α-crystallin accumulate that can reduce chaperone function; this is compounded in cataract lenses. Extensive in vitro glycation, carbamylation and oxidation all decrease chaperone ability. In the present study, we report the effect of the modifiers malondialdehyde, acetaldehyde and methylglyoxal, all of which are pertinent to cataract. Also modification by aspirin, which is known to delay cataract and other diseases, has been investigated. Recently, two point mutations of arginine residues were shown to cause congenital cataract. 1,2-Cyclohexanedione modifies arginine residues, and the extent of modification needed for a change in chaperone function was investigated. Only methylglyoxal and extensive modification by 1,2-cyclohexanedione caused a decrease in chaperone function. This highlights the robust nature of α-crystallin.

scholarly journals Bifunctional Chloroplastic DJ-1B from Arabidopsis thaliana is an Oxidation-Robust Holdase and a Glyoxalase Sensitive to H2O2

Antioxidants ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 8 ◽  
Author(s):  
Aleksandra Lewandowska ◽  
Trung Nghia Vo ◽  
Thuy-Dung Ho Nguyen ◽  
Khadija Wahni ◽  
Didier Vertommen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Arabidopsis Thaliana ◽  
Secondary Structure ◽  
Structure And Function ◽  
Transcript Levels ◽  
Chaperone Function ◽  
Multifunctional Enzymes ◽  
And Function ◽  
Discrete Functions

Members of the DJ-1 protein family are multifunctional enzymes whose loss increases the susceptibility of the cell to oxidative stress. However, little is known about the function of the plant DJ-1 homologs. Therefore, we analyzed the effect of oxidation on the structure and function of chloroplastic AtDJ-1B and studied the phenotype of T-DNA lines lacking the protein. In vitro oxidation of AtDJ-1B with H2O2 lowers its glyoxalase activity, but has no effect on its holdase chaperone function. Remarkably, upon oxidation, the thermostability of AtDJ-1B increases with no significant alteration of the overall secondary structure. Moreover, we found that AtDJ-1B transcript levels are invariable, and loss of AtDJ-1B does not affect plant viability, growth and stress response. All in all, two discrete functions of AtDJ-1B respond differently to H2O2, and AtDJ-1B is not essential for plant development under stress.

scholarly journals The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

1996 ◽  
Vol 135 (5) ◽  
pp. 1229-1237 ◽  
Author(s):  
G L Bush ◽  
D I Meyer
Keyword(s):  
Protein Translocation ◽  
In Vitro Assay ◽  
Secretory Proteins ◽  
Vitro Assay ◽  
Chaperone Function ◽  
Cotranslational Folding ◽  
Yeast Lysate ◽  
Shock Proteins ◽  
First Time

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.

Dual Inhibition of Bcr-Abl and Hsp90 By C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 914-914
Author(s):  
Lixian Wu ◽  
Jing Yu ◽  
Yang Liu ◽  
Lou Liguang ◽  
Yong Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Conflicts Of Interest ◽  
Chaperone Function ◽  
Abl Kinase ◽  
Fluorescence Measurements ◽  
Biochemical Assays ◽  
Imatinib Resistant ◽  
Hsp90 Chaperone ◽  
Hsp90 Function

Abstract Purpose: Although such tyrosine kinase inhibitors (TKIs) as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of CML patients, TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation and immunoblotting were applied to examine Hsp90 function; Colony-forming unit (CFU), long-term culture-initiating cells (LTC-ICs), and flow cytometry were used to test CML progenitor and stem cells. Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, inhibited the growth of both imatinib-sensitive and resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38- cells in vitro. Moreover, C086 could decreased the number of CD45+, CD45+CD34+CD38+ and CD45+CD34+CD38- cells in CML NOD-SCID mice. Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enzyme activity after resealing within ghost erythrocyte cells, and protection by α-crystallin against fructose-induced inactivation

2002 ◽  
Vol 368 (3) ◽  
pp. 865-874 ◽  
Author(s):  
Barry K. DERHAM ◽  
John J. HARDING
Keyword(s):  
Enzyme Activity ◽  
Molecular Chaperone ◽  
Soluble Fraction ◽  
Erythrocyte Ghosts ◽  
Ghost Cell ◽  
Chaperone Function ◽  
Zero Time ◽  
Soluble Fractions

The role of α-crystallin as a molecular chaperone has been shown in many in vitro studies. In the present paper, we report on the chaperone function of α-crystallin within resealed erythrocyte ghosts. Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24h. The ghost cell suspension was separated into soluble and membrane fractions. Five of the enzymes had significantly greater enzyme activity after 24h than the control within the soluble fractions. Fructation caused a decrease in enzyme activity (relative to the control). Resealing of α-crystallin within the ghost cell alongside the enzymes protected against inactivation by fructose within the soluble fraction.

scholarly journals The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

2000 ◽  
Vol 20 (19) ◽  
pp. 7146-7159 ◽  
Author(s):  
Dick D. Mosser ◽  
Antoine W. Caron ◽  
Lucie Bourget ◽  
Anatoli B. Meriin ◽  
Michael Y. Sherman ◽  
...  
Keyword(s):  
Protective Effects ◽  
Atpase Domain ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Mutant Proteins ◽  
Chaperone Function ◽  
Induced Apoptosis ◽  
Jnk Activation ◽  
In Cells

ABSTRACT Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochromec-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

Phase 1 Clinical Trial of KOS-953 + Bortezomib (BZ) in Relapsed Refractory Multiple Myeloma (MM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 362-362 ◽  
Author(s):  
Asher Alban Chanan-Khan ◽  
P. G. Richardson ◽  
M. Alsina ◽  
M. Carroll ◽  
S. Lonial ◽  
...  
Keyword(s):  
Stress Response ◽  
Dose Escalation ◽  
Phase 1 ◽  
Single Agent ◽  
Annexin V ◽  
20S Proteasome ◽  
Chaperone Function ◽  
Hepatic Involvement ◽  
Hsp90 Chaperone

Abstract Background: KOS-953 is a novel formulation of 17-AAG, an inhibitor of HSP90 chaperone function. Disruption of specific heteroprotein complexes results in their decreased stability, impaired cellular trafficking and proteasomal degradation. KOS-953 shows in vitro activity against MM cells from patients (pts) resistant to BZ and extends survival of MM mice models. Treatment of MM cells with BZ triggers significant HSP90 up-regulation as a stress response; KOS-953 blocks this stress response, thereby enhancing MM cell sensitivity to BZ. Objectives: To define the recommended dose of KOS 953 and BZ in pts with relapsed and relapsed/refractory MM; to evaluate the PK of KOS-953 and its active metabolite in combination with BZ, and to evaluate biological activity of the combination. Methods: Pts receive IV therapy (BZ followed by 1-hr infusion KOS-953) twice weekly for 2 out of 3 weeks. Dose escalation of both agents in a step-wise manner occurs (BZ: 0.7, 1.0 and 1.3 mg/m2; KOS-953 100, 150 and 220 mg/m2). PK and PD are performed following the 1st and 4th infusion. KOS-953 and its metabolite are quantified in plasma. Bone marrow aspirates (BM) are examined for apoptosis, proliferation, pAKT, and change in HSP, IL-6 and IGF-R1 expression; peripheral blood leucocytes are examined for change in HSP70/90 levels and proteasome 20S function. EMBT criteria are used to assess response. Results: 15 pts (8F; median age 61 yrs; median prior regimens 4; 11 BZ-refractory and 9 with prior SCT)) enrolled in 4 dose levels (DLs 1–4). Dose limiting toxicity (G4 hepatotoxicity) was observed in 1 pt at DL#3 with hepatic involvement with MM and congestive heart failure (secondary to cardiac amyloidosis) who subsequently died of heart disease. No further toxicity was seen in the 5 other pts at this dose level. Enrollment to DL#4 is underway (BZ 1.3 mg/m2; KOS-953 150 mg/m2); no other G≥3 drug-related toxicity has been seen. G1-2 drug-related toxicity: diarrhea (n=5), fatigue (n=5), thrombocytopenia (n=3), infusional reactions (n=4), increased AST (n=3), anemia (n=3) and rash (n=3). Comparison of PK data for parent and metabolite to the single agent data showed similar kinetics (clearance remained unchanged at 48.9 ± 16.6 L/hr). Ratio of exposure comparing metabolite to parent was 96.5% ± 90.5. Evaluation of 20S proteasome function showed a 40% decrease at BZ (0.7 and 1.0 mg/m2) at the end of infusion on Day 11 (similar to predicted). Signs of antitumor activity: 1 BZ-refractory pt with SD x 6 cycles at DL#1; at DL#2, 2 BZ-refractory pts with MR after 9 cycles and one BZ-naive pt with MR (100% reduction of urine M-protein; 41% reduction in serum IgA x 9 cycles); and DL#3 1 BZ-refractory pt with MR x 6+ cycles. PBLs showed increased apoptosis by Annexin V (P=0.02). Conclusions: Dose escalation continues to define the optimal doses of the combination. No additive toxicity has been observed; no adverse PK interactions are observed at doses tested to date. Similar inhibition of the 20S proteasome is observed compared to historical BZ data. Encouraging activity has been seen in both BZ-naive and heavily treated, BZ-refractory MM pts.

scholarly journals Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum

2015 ◽  
Vol 112 (7) ◽  
pp. E616-E624 ◽  
Author(s):  
Filipa Teixeira ◽  
Helena Castro ◽  
Tânia Cruz ◽  
Eric Tse ◽  
Philipp Koldewey ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Protein Unfolding ◽  
Protozoan Parasite ◽  
Chaperone Activity ◽  
Dual Function ◽  
Active Site Cysteine ◽  
Chaperone Function ◽  
Client Proteins

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx’s ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx’s chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

scholarly journals Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 5024-5033 ◽  
Author(s):  
Yongchao Wang ◽  
Warren Fiskus ◽  
Daniel G. Chong ◽  
Kathleen M. Buckley ◽  
Kavita Natarajan ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Heat Shock Protein 90 ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transducers ◽  
Downstream Signaling ◽  
Jak2 Inhibitor ◽  
Chaperone Function ◽  
Mitogen Activated Protein ◽  
Induced Apoptosis

AbstractThe mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL–negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34+ MPN cells than normal CD34+ HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

scholarly journals Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells

2007 ◽  
Vol 407 (3) ◽  
pp. 407-417 ◽  
Author(s):  
Anton L. Bryantsev ◽  
Svetlana Yu. Kurchashova ◽  
Sergey A. Golyshev ◽  
Vladimir Yu. Polyakov ◽  
Herman F. Wunderink ◽  
...  
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Firefly Luciferase ◽  
Negative Regulator ◽  
Unfolded Proteins ◽  
Reporter Protein ◽  
Chaperone Function ◽  
Hsp27 Phosphorylation

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

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