adenofection-method-for-studying-role-molecular-chaperones-cellular

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H(2)O(2), induction by heat shock was not antagonized by pretreatment with the antioxidant N-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathione S-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation.

Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1.

Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.

c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade.

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an alpha-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.

The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27.

A familial form of desmin-related myopathy (DRM) is associated with a missense mutation (R120G) in alphaB-crystallin (alphaB) and is characterized by intracellular desmin aggregation. Because alphaB is a molecular chaperone that participates in the assembly of desmin filaments, it has been suggested that the desmin aggregation might be due to the loss of alphaB function. We report here that alphaBR120G has indeed impaired in vivo function and structure as reflected by a highly reduced capacity to protect cells against heat shock and by an abnormal supramolecular organization even in cells not expressing desmin. In many cells, alphaBR120G accumulated in inclusion bodies that had characteristics of aggresomes concentrating around the centrosome following a microtubule-facilitated process. Three distinct chaperone mechanisms could reduce or even prevent the formation of the alphaBR120G aggresomes. Wild-type alphaB and Hsp27 prevented aggresome formation by co-oligomerizing with alphaBR120G. Hsp70 with its co-chaperone Hdj-1 or Chip-1 but not a mutant of Chip-1 lacking ubiquitin ligase activity, reduced the frequency of aggresome formation likely by targeting alphaBR120G for degradation. Finally, HspB8 interacted only transiently with alphaB but nonetheless rescued the alphaBR120G oligomeric organization, suggesting that it acted as a true chaperone assisting in the folding of the mutant protein. Hence, the formation of inclusion bodies in alphaBR120G-mediated DRM is probably due to the misfolding of alphaBR120G per se and can be delayed or prevented by expression of the wild type alphaB allele or other molecular chaperones, thereby explaining the adult onset of the disease.

Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alphaB-crystallin mutant.

Eine famili&auml;re Form der Desminopathie (DRM) ist eine Missense-Mutation (R120G) in AlphaB-Crystallin (AlphaB) zugeordnet und zeichnet sich durch intrazellul&auml;re Desmin Aggregation. Denn AlphaB eine molekulare Chaperone, die an der Versammlung des Desmin-Filamente beteiligt ist, wurde es vermutet, dass die Desmin-Aggregation durch den Verlust der AlphaB Funktion m&ouml;glicherweise. Wir berichten hier, dass die alphaBR120G tats&auml;chlich in-vivo-Funktion und Struktur beeintr&auml;chtigt ist wie durch eine stark reduzierte F&auml;higkeit zum Schutz der Zellen gegen W&auml;rmeschock und durch eine St&ouml;rung der supramolekularen Organisation auch in Zellen nicht auszudr&uuml;cken Desmin wider. In vielen Zellen alphaBR120G im Einschlussk&ouml;rperchen, die Merkmale der Aggresomes konzentriert sich rund um das Zentrosom nach einem Microtubule-erleichtert hatte gesammelt. Drei unterschiedliche Anstandsdame Mechanismen k&ouml;nnten reduzieren oder sogar verhindern die Bildung von alphaBR120G-Aggresomes. Wildtyp AlphaB und Hsp27 verhindert Aggresome Bildung von co-oligomerizing mit alphaBR120G. Hsp70 mit seiner Co-chaperone Hdj-1 oder Chip-1 aber keine Mutante der Chip-1 fehlt Ubiquitin Ligase T&auml;tigkeit, reduziert die H&auml;ufigkeit der Aggresome-Formation, die wahrscheinlich durch gezielte alphaBR120G auf Leistungsabfall. Schlie&szlig;lich HspB8 interagiert nur vor&uuml;bergehend mit AlphaB aber dennoch rettete die alphaBR120G Oligomere Organisation, was darauf hindeutet, dass es als eine wahre Anstandsdame, die Unterst&uuml;tzung bei der Faltung des mutierten Proteins gehandelt. Daher kann die Bildung von Einschlussk&ouml;rperchen in alphaBR120G-vermittelte DRM ist wahrscheinlich auf die misfolding der alphaBR120G schlechthin und verz&ouml;gert oder durch Auspr&auml;gung der Wildtyp-Allel AlphaB oder andere Molekulare Chaperone, erkl&auml;ren damit die adulte Form der Krankheit verhindert.

Unterschiedliche Anstandsdame Mechanismen k&ouml;nnen die Bildung von Aggresomes durch die Myopathie verursachender R120G AlphaB-Crystallin Mutant verz&ouml;gern.

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

The family of small heat shock proteins (sHsp) is composed of 10 members in mammals, four of which are found mutated in diseases associated with the accumulation of protein aggregates. Though many sHsp have demonstrated molecular chaperone activity in vitro in cell-free conditions, their activity in vivo in the normal cellular context remains unclear. In the present study, we investigated the capacity of the sHsp, HspB8/Hsp22, to prevent protein aggregation in the cells using the polyglutamine protein Htt43Q as a model. In control conditions, Htt43Q accumulated in perinuclear inclusions composed of SDS-insoluble aggregates. Co-transfected with Htt43Q, HspB8 became occasionally trapped within the inclusions; however, in most cells, HspB8 blocked inclusion formation. Biochemical analyses indicated that HspB8 inhibited the accumulation of SDS-insoluble Htt43Q as efficiently as Hsp40 which was taken as a positive control. Htt43Q then accumulated in the SDS-soluble fraction, provided that protein degradation was blocked by proteasome and autophagy inhibitors. In contrast, the other sHsp Hsp27/HspB1 and alphaB-crystallin/HspB5 had no effect. This suggested that HspB8 functions as a molecular chaperone, maintaining Htt43Q in a soluble state competent for rapid degradation. Analyses of Hsp27-HspB8 chimeric proteins indicated that the C-terminal domain of HspB8 contains the specific sequence necessary for chaperone activity. Missense mutations in this domain at lysine 141, which are found in human motor neuropathies, significantly reduced the chaperone activity of the protein. A decrease in the HspB8 chaperone activity may therefore contribute to the development of these diseases.

HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells.

Mutations in the alpha-crystallin domain of 4 of the small heat shock proteins (sHsp) (Hsp27/HspB1, alphaA-crystallin/ HspB4, alphaB-crystallin/HspB5, and HspB8) are responsible for dominant inherited diseases in humans. One such mutation at a highly conserved arginine residue was shown to cause major conformational defects and intracellular aggregation of alphaA- and alphaB-crystallins and HspB8. Here, we studied the effect of this Arg mutation on the structure and function of Hsp27. Chinese hamster Hsp27 with Arg148 replaced by Gly (Hsp27R148G) formed dimers in vitro and in vivo, which contrasted with the 12- or 24-subunit oligomers formed by the wild-type protein (Hsp27WT). Despite these alterations, Hsp27R148G had a chaperone activity almost as high as Hsp27WT. The dimers of Hsp27R148G did not further deoligomerize on phosphorylation and like the dimers formed by phosphorylated Hsp27WT were not affected by the deletion of the N-terminal WD/EPF (single letter amino acid code) motif, suggesting that mutation of Arg148, deletion of the N-terminal WD/EPF motif, and phosphorylation of Ser90 may produce similar structural perturbations. Nevertheless, the structure of Hsp27R148G appeared unstable, and the mutated protein accumulated as aggregates in many cells. Both a lower basal level of phosphorylation of Hsp27R148G and the coexpression of Hsp27WT could reduce the frequency of formation of these aggregates, suggesting possible mechanisms regulating the onset of the sHsp-mediated inherited diseases.

Structural instability caused by a mutation at a conserved arginine in the alpha-crystallin domain of Chinese hamster heat shock protein 27.

Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy.

HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy.

Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8.Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as ATP-independent molecular chaperones that can bind unfolded proteins and facilitate their processing via the help of ATP-dependent chaperones such as the Hsp70 machine, in which Bag3 may act as a molecular link between HspB, Hsp70, and the ubiquitin ligases. However, here we show that HspB8 and Bag3 act in a non-canonical manner unrelated to the classical chaperone model. Rather, HspB8 and Bag3 induce the phosphorylation of the alpha-subunit of the translation initiator factor eIF2, which in turn causes a translational shut-down and stimulates autophagy. This function of HspB8.Bag3 does not require Hsp70 and also targets fully folded substrates. HspB8.Bag3 activity was independent of the endoplasmic reticulum (ER) stress kinase PERK, demonstrating that its action is unrelated to ER stress and suggesting that it activates stress-mediated translational arrest and autophagy through a novel pathway.

HspB8 participates in protein quality control by a non-chaperone-like mechanism that requires eIF2{alpha} phosphorylation.

The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated athanogene 3). In the present study we identify the binding regions in HspB8 and Bag3 crucial for their interaction. We present evidence that HspB8 binds to Bag3 through the hydrophobic groove formed by its strands beta4 and beta8, a region previously known to be responsible for the formation and stability of higher-order oligomers of many sHsps (small Hsps). Moreover, we demonstrate that two conserved IPV (Ile-Pro-Val) motifs in Bag3 mediate its binding to HspB8 and that deletion of these motifs suppresses HspB8 chaperone activity towards mutant Htt43Q (huntingtin exon 1 fragment with 43 CAG repeats). In addition, we show that Bag3 can bind to the molecular chaperone HspB6. The interaction between HspB6 and Bag3 requires the same regions that are involved in the HspB8-Bag3 association and HspB6-Bag3 promotes clearance of aggregated Htt43Q. Our findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their substrate proteins for degradation. Interestingly, a mutation in one of Bag3 IPV motifs has recently been associated with the development of severe dominant childhood muscular dystrophy, suggesting a possible important physiological role for HspB-Bag3 complexes in this disease.

Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction.

Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.

Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve.

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

Article	Year
Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1. The Journal of biological chemistry\| PubMed ID: 12077134	2002
c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade. The Biochemical journal\| PubMed ID: 12646044	2003
The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27. FEBS letters\| PubMed ID: 12804778	2003
Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alphaB-crystallin mutant. Human molecular genetics\| PubMed ID: 12812987	2003
Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27. The Journal of biological chemistry\| PubMed ID: 15033973	2004
HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells. Human molecular genetics\| PubMed ID: 15879436	2005
Structural instability caused by a mutation at a conserved arginine in the alpha-crystallin domain of Chinese hamster heat shock protein 27. Cell stress & chaperones\| PubMed ID: 16038412	2005
HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy. The Journal of biological chemistry\| PubMed ID: 18006506	2008
HspB8 participates in protein quality control by a non-chaperone-like mechanism that requires eIF2{alpha} phosphorylation. The Journal of biological chemistry\| PubMed ID: 19114712	2009
Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction. The Biochemical journal\| PubMed ID: 19845507	2010
Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve. Journal of immunological methods\| PubMed ID: 20035759	2010
A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis. PLoS genetics\| PubMed ID: 26496431	2015

Herman Lambert

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