共查询到20条相似文献,搜索用时 31 毫秒
1.
Kitamura A Kubota H Pack CG Matsumoto G Hirayama S Takahashi Y Kimura H Kinjo M Morimoto RI Nagata K 《Nature cell biology》2006,8(10):1163-1170
Polyglutamine (polyQ)-expansion proteins cause neurodegenerative disorders including Huntington's disease, Kennedy's disease and various ataxias. The cytotoxicity of these proteins is associated with the formation of aggregates or other conformationally toxic species. Here, we show that the cytosolic chaperonin CCT (also known as TRiC) can alter the course of aggregation and cytotoxicity of huntingtin (Htt)-polyQ proteins in mammalian cells. Disruption of the CCT complex by RNAi-mediated knockdown enhanced Htt-polyQ aggregate formation and cellular toxicity. Analysis of the aggregation states of the Htt-polyQ proteins by fluorescence correlation spectroscopy revealed that CCT depletion results in the appearance of soluble Htt-polyQ aggregates. Similarly, overexpression of all eight subunits of CCT suppressed Htt aggregation and neuronal cell death. These results indicate that CCT has an essential role in protecting against the cytotoxicity of polyQ proteins by affecting the course of aggregation. 相似文献
2.
Behrends C Langer CA Boteva R Böttcher UM Stemp MJ Schaffar G Rao BV Giese A Kretzschmar H Siegers K Hartl FU 《Molecular cell》2006,23(6):887-897
Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model. In vitro reconstitution experiments revealed that TRiC, in cooperation with the Hsp70 system, promotes the assembly of polyQ-expanded fragments of huntingtin (Htt) into soluble oligomers of approximately 500 kDa. Similar oligomers were observed in yeast cells upon TRiC overexpression and were found to be benign, in contrast to conformationally distinct Htt oligomers of approximately 200 kDa, which accumulated at normal TRiC levels and correlated with inhibition of cell growth. We suggest that TRiC cooperates with the Hsp70 system as a key component in the cellular defense against amyloid-like protein misfolding. 相似文献
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Sangjune Kim Do-Young Park Dohyun Lee Wanil Kim Young-Hun Jeong Juhyun Lee Sung-Kee Chung Hyunjung Ha Bo-Hwa Choi Kyong-Tai Kim 《Molecular and cellular biology》2014,34(4):643-652
Misfolding of proteins containing abnormal expansions of polyglutamine (polyQ) repeats is associated with cytotoxicity in several neurodegenerative disorders, including Huntington''s disease. Recently, the eukaryotic chaperonin TRiC hetero-oligomeric complex has been shown to play an important role in protecting cells against the accumulation of misfolded polyQ protein aggregates. It is essential to elucidate how TRiC function is regulated to better understand the pathological mechanism of polyQ aggregation. Here, we propose that vaccinia-related kinase 2 (VRK2) is a critical enzyme that negatively regulates TRiC. In mammalian cells, overexpression of wild-type VRK2 decreased endogenous TRiC protein levels by promoting TRiC ubiquitination, but a VRK2 kinase-dead mutant did not. Interestingly, VRK2-mediated downregulation of TRiC increased aggregate formation of a polyQ-expanded huntingtin fragment. This effect was ameliorated by rescue of TRiC protein levels. Notably, small interference RNA-mediated knockdown of VRK2 enhanced TRiC protein stability and decreased polyQ aggregation. The VRK2-mediated reduction of TRiC protein levels was subsequent to the recruitment of COP1 E3 ligase. Among the members of the COP1 E3 ligase complex, VRK2 interacted with RBX1 and increased E3 ligase activity on TRiC in vitro. Taken together, these results demonstrate that VRK2 is crucial to regulate the ubiquitination-proteosomal degradation of TRiC, which controls folding of polyglutamine proteins involved in Huntington''s disease. 相似文献
5.
Sangjune Kim Dohyun Lee Juhyun Lee Haengjin Song Hyo-Jin Kim Kyong-Tai Kim 《Molecular and cellular biology》2015,35(10):1754-1762
Molecular chaperones monitor the proper folding of misfolded proteins and function as the first line of defense against mutant protein aggregation in neurodegenerative diseases. The eukaryotic chaperonin TRiC is a potent suppressor of mutant protein aggregation and toxicity in early stages of disease progression. Elucidation of TRiC functional regulation will enable us to better understand the pathological mechanisms of neurodegeneration. We have previously shown that vaccinia-related kinase 2 (VRK2) downregulates TRiC protein levels through the ubiquitin-proteasome system by recruiting the E3 ligase COP1. However, although VRK2 activity was necessary in TRiC downregulation, the phosphorylated substrate was not determined. Here, we report that USP25 is a novel TRiC interacting protein that is also phosphorylated by VRK2. USP25 catalyzed deubiquitination of the TRiC protein and stabilized the chaperonin, thereby reducing accumulation of misfolded polyglutamine protein aggregates. Notably, USP25 deubiquitinating activity was suppressed when VRK2 phosphorylated the Thr680, Thr727, and Ser745 residues. Impaired USP25 deubiquitinating activity after VRK2-mediated phosphorylation may be a critical pathway in TRiC protein destabilization. 相似文献
6.
Oksana A. Sergeeva Bo Chen Cameron Haase-Pettingell Steven J. Ludtke Wah Chiu Jonathan A. King 《The Journal of biological chemistry》2013,288(24):17734-17744
Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits. 相似文献
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Zhiqiang Zheng Aimin Li Brandon B. Holmes Jayne C. Marasa Marc I. Diamond 《The Journal of biological chemistry》2013,288(9):6063-6071
Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity. 相似文献
9.
Joan S Steffan 《Cell cycle (Georgetown, Tex.)》2010,9(17):3401-3413
The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.1 In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases. 相似文献
10.
Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1–8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells. 相似文献
11.
ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation 总被引:2,自引:0,他引:2
Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, HA-1077, and H-1152P), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. 相似文献
12.
The role of molecular chaperones in human misfolding diseases 总被引:1,自引:0,他引:1
Sarah A. Broadley 《FEBS letters》2009,583(16):2647-144
Human misfolding diseases arise when proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. Molecular chaperones, such as Hsp70 and TCP-1 Ring Complex (TRiC)/chaperonin containing TCP-1 (CCT), have been implicated as potent modulators of misfolding disease. These chaperones suppress toxicity of disease proteins and modify early events in the aggregation process in a cooperative and sequential manner reminiscent of their functions in de novo protein folding. Further understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles that could potentially be exploited for therapeutic purposes. 相似文献
13.
The ring-shaped hetero-oligomeric chaperonin TRiC/CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins. 相似文献
14.
Knee KM Goulet DR Zhang J Chen B Chiu W King JA 《Protein science : a publication of the Protein Society》2011,20(1):30-41
Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT. 相似文献
15.
The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containing TCP-1) is a large (approximately 900 kDa) multisubunit complex that mediates protein folding in the eukaryotic cytosol. The physiological substrate spectrum of TRiC is still poorly defined. Genetic and biochemical data show that it is required for the folding of the cytoskeletal proteins actin and tubulin. Recent years have witnessed a steady stream of reports that describe other proteins that require TRiC for proper folding. Furthermore, analysis of the transit of newly synthesized proteins through TRiC in intact cells suggests that the chaperonin contributes to the folding of a distinct subset of cellular proteins. Here we review the current understanding of a role for TRiC in the folding of newly synthesized polypeptides, with a focus on some of the individual proteins that require TRiC. 相似文献
16.
Ernesto Llamas Salvador TorresMontilla Hyun Ju Lee María Victoria Barja Elena Schlimgen Nick Dunken Prerana Wagle Wolfgang Werr Alga Zuccaro Manuel RodríguezConcepcin David Vilchez 《Aging cell》2021,20(8)
The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T‐complex protein‐1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change. 相似文献
17.
Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction. 相似文献
18.
Swinnen E Büttner S Outeiro TF Galas MC Madeo F Winderickx J Franssens V 《Biochemical Society transactions》2011,39(5):1476-1481
PD (Parkinson's disease) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra, which affects an increasing number of the elderly population worldwide. One of the major hallmarks of PD is the occurrence of intracellular protein deposits in the dying neurons, termed Lewy bodies, which contain different proteins, including aggregated α-synuclein and its interacting protein synphilin-1. During the last decade, a number of groups developed yeast models that reproduced important features of PD and allowed the deciphering of pathways underlying the cytotoxicity triggered by α-synuclein. Here, we review the recent contributions obtained with yeast models designed to study the presumed pathobiology of synphilin-1. These models pointed towards a crucial role of the sirtuin Sir2 and the chaperonin complex TRiC (TCP-1 ring complex)/CCT (chaperonin containing TCP-1) in handling misfolded and aggregated proteins. 相似文献
19.
The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo. 相似文献
20.
Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus 总被引:10,自引:0,他引:10
Gafni J Hermel E Young JE Wellington CL Hayden MR Ellerby LM 《The Journal of biological chemistry》2004,279(19):20211-20220
Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis. 相似文献