RESUMO
Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging.
Assuntos
Envelhecimento , Antígenos CD/metabolismo , Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans , Drosophila melanogaster , Endocitose , Humanos , Longevidade , Lisossomos/metabolismo , Proteólise , Proteoma , Transdução de Sinais , Somatomedinas , UbiquitinaçãoRESUMO
Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.
Assuntos
Dobramento de Proteína , Proteostase , Sobrevivência Celular , Proteoma/metabolismo , Estresse MecânicoRESUMO
Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas dos Microfilamentos/genética , Músculo Esquelético/metabolismo , Estresse Mecânico , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/genética , Actinina/genética , Actinas/genética , Animais , Autofagia/genética , Linhagem Celular , Citoesqueleto/genética , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Estriado/metabolismo , Miofibrilas/genética , Miofibrilas/metabolismo , Domínios PDZ/genética , Isoformas de Proteínas/genética , Sinaptofisina/genéticaRESUMO
BACKGROUND: Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS: We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS: Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS: We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.
Assuntos
Filaminas/genética , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/patologia , Proteômica/métodos , Estresse Fisiológico/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/genética , Humanos , Camundongos , Podócitos/metabolismo , Proteinúria/genética , Proteinúria/fisiopatologia , Distribuição Aleatória , RatosRESUMO
Skeletal muscle is a target of contraction-induced loading (CiL), leading to protein unfolding or cellular perturbations, respectively. While cytoskeletal desmin is responsible for ongoing structural stabilization, in the immediate response to CiL, alpha-crystallin B (CRYAB) is phosphorylated at serine 59 (pCRYABS59) by P38, acutely protecting the cytoskeleton. To reveal adaptation and deadaptation of these myofibrillar subsystems to CiL, we examined CRYAB, P38, and desmin regulation following resistance exercise at diverse time points of a chronic training period. Mechanosensitive JNK phosphorylation (pJNKT183/Y185) was determined to indicate the presence of mechanical components in CiL. Within 6 wk, subjects performed 13 resistance exercise bouts at the 8-12 repetition maximum, followed by 10 days detraining and a final 14th bout. Biopsies were taken at baseline and after the 1st, 3rd, 7th, 10th, 13th, and 14th bout. To assess whether potential desensitization to CiL can be mitigated, one group trained with progressive and a second with constant loading. As no group differences were found, all subjects were combined for statistics. Total and phosphorylated P38 was not regulated over the time course. pCRYABS59 and pJNKT183/Y185 strongly increased following the unaccustomed first bout. This exercise-induced pCRYABS59/pJNKT183/Y185 increase disappeared with the 10th until 13th bout. As response to the detraining period, the 14th bout led to a renewed increase in pCRYABS59. Desmin content followed pCRYABS59 inversely, i.e., was up- when pCRYABS59 was downregulated and vice versa. In conclusion, the pCRYABS59 response indicates increase and decrease in resistance to CiL, in which a reinforced desmin network could play an essential role by structurally stabilizing the cells.
Assuntos
Adaptação Fisiológica/genética , Desmina/genética , Músculo Esquelético/metabolismo , Cadeia B de alfa-Cristalina/genética , Adulto , Citoesqueleto/genética , Citoesqueleto/metabolismo , Desmina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Fosforilação/genética , Treinamento Resistido/efeitos adversos , Adulto Jovem , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Cellular and organismal survival depend on the ability to maintain the proteome, even under conditions that threaten protein integrity. BCL2-associated athanogene 3 (BAG3) is essential for protein homeostasis (proteostasis) in stressed cells. Owing to its multi-domain structure, it engages in diverse processes that are crucial for proteome maintenance. BAG3 promotes the activity of molecular chaperones, sequesters and concentrates misfolded proteins, initiates autophagic disposal, and balances transcription, translation and degradation. In this Cell Science at a Glance article and the accompanying poster, we discuss the functions of this multi-functional proteostasis tool with a focus on mechanical stress protection and describe the importance of BAG3 for human physiology and pathophysiology.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteostase , Animais , Autofagia , Citoesqueleto/metabolismo , Filaminas/metabolismo , Humanos , Chaperonas Moleculares/metabolismoRESUMO
The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Complexos Multiproteicos/genética , Músculo Esquelético/metabolismo , Miócitos de Músculo Liso/metabolismo , Estresse Mecânico , Serina-Treonina Quinases TOR/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Fenômenos Biomecânicos , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Filaminas/genética , Filaminas/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Músculo Esquelético/citologia , Miócitos de Músculo Liso/ultraestrutura , Ligação Proteica , Biossíntese de Proteínas , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Genetic studies have linked the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in the etiology of schizophrenia and other psychiatric disorders. However, the function of the protein encoded by this locus, LG72, is currently controversially discussed. Some studies have suggested that LG72 binds to and regulates the activity of the peroxisomal enzyme D-amino-acid-oxidase, while others proposed an alternative role of this protein due to its mitochondrial location in vitro. Studies with transgenic mice expressing LG72 further suggested that high levels of LG72 lead to an impairment of mitochondrial functions with a concomitant increase in reactive oxygen species production. In the present study, we now performed extensive interaction analyses and identified the mitochondrial methionine-R-sulfoxide reductase B2 (MSRB2) as a specific interaction partner of LG72. MSRB2 belongs to the MSR protein family and functions in mitochondrial oxidative stress defense. Based on our results, we propose that LG72 is involved in the regulation of mitochondrial oxidative stress.
Assuntos
Proteínas de Transporte/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas dos Microfilamentos , Ligação ProteicaRESUMO
Proteome maintenance in contracting skeletal and cardiac muscles depends on the chaperone-regulating protein BAG3. Reduced BAG3 activity leads to muscle weakness and heart failure in animal models and patients. BAG3 and its chaperone partners recognize mechanically damaged muscle proteins and initiate their disposal through chaperone-assisted selective autophagy (CASA). However, molecular details of the force-dependent regulation of BAG3 have remained elusive so far. Here, we demonstrate that mechanical stress triggers the dephosphorylation of BAG3 in human muscle and in isolated cells. We identify force-regulated phospho-switches in BAG3 that control CASA complex assembly and CASA activity. Differential proteomics reveal RAB GTPases, which organize membrane traffic and fusion, as dephosphorylation-dependent interactors of BAG3. In fact, RAB7A and RAB11B are shown here to be essential for CASA in skeletal muscle cells. Moreover, BAG3 dephosphorylation is also observed upon induction of mitophagy, suggesting an involvement of the cochaperone in the RAB7A-dependent autophagic engulfment of damaged mitochondria in exercised muscle. Cooperation of BAG3 with RAB7A relies on a direct interaction of both proteins, which is regulated by the nucleotide state of the GTPase and by association with the autophagosome membrane protein LC3B. Finally, we provide evidence that BAG3 and RAB7A also cooperate in non-muscle cells and propose that overactivation of CASA in RAB7A-L129F patients contributes to the loss of peripheral neurons in Charcot-Marie-Tooth neuropathy.
RESUMO
Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there is a noticeably diminished efficiency of both the ubiquitin-proteasome system and autophagy that impairs the muscle capacity to prevent the formation or mediate the degradation of aggregates. Transfection studies of cultured muscle cells imitate events observed in the patient's affected muscle and therefore provide a helpful model for testing future therapeutic strategies.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Mutação , Fenótipo , Actinas/metabolismo , Adulto , Proteínas Contráteis/genética , Progressão da Doença , Feminino , Filaminas , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Linhagem , Ligação Proteica , Proteólise , UbiquitinaçãoRESUMO
The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Chaperonas Moleculares/metabolismo , Ricina/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Conformação Proteica , Ribossomos/metabolismo , Ricina/toxicidade , UbiquitinaçãoRESUMO
Basically, all mammalian tissues are constantly exposed to a variety of environmental mechanical signals. Depending on the signal strength, mechanics intervenes in a multitude of cellular processes and is thus capable of inducing simple cellular adaptations but also complex differentiation processes and even apoptosis. The underlying recognition typically depends on mechanosensitive proteins, which most often sense the mechanical signal for the induction of a cellular signaling cascade by changing their protein conformation. However, the fate of mechanosensors after mechanical stress application is still poorly understood, and it remains unclear whether protein degradation pathways affect the mechanosensitivity of cells. Here, we show that cyclic stretch induces autophagosome formation in a time-dependent manner. Formation depends on the cochaperone BAG family molecular chaperone regulator 3 (BAG3) and thus likely involves BAG3-mediated chaperone-assisted selective autophagy. Furthermore, we demonstrate that strain-induced cell reorientation is clearly delayed upon inhibition of autophagy, suggesting a bidirectional cross-talk between mechanotransduction and autophagic degradation. The strength of the observed delay depends on stable adhesion structures and stress fiber formation in a Ras homologue family member A (RhoA)-dependent manner.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Mecanorreceptores/metabolismo , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Autofagia/fisiologia , Fenômenos Biomecânicos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Mecanorreceptores/citologia , Mecanotransdução Celular , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Proteólise , Ratos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Animais , Autofagia , Sítios de Ligação , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/terapia , Cardiomiopatia Restritiva/terapia , Criança , Modelos Animais de Doenças , Regulação da Expressão Gênica , Terapia Genética , Coração , Proteínas de Choque Térmico , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Proteômica , Sarcômeros/metabolismoRESUMO
Keratin (K) intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which leads to a large number of human disorders. KRT5 or KRT14 mutations cause epidermolysis bullosa simplex (EBS). The considerable intra- and interfamilial variability in EBS suggests modifying loci, most of which are unknown. In many human disorders, chaperones and the ubiquitin-proteasome system have been found to modify disease severity, thereby providing novel therapy targets. Here, we demonstrate upregulation of stress-induced Hsp70 and Hsp90 in two EBS models, namely, in neonatal K5(-/-) mice and upon proteasome inhibition in cells that stably express the disease-causing mutation K14-p.Arg125Cys, both harboring keratin aggregates. Furthermore, proteasome inhibition caused nuclear translocation of pHSF-1 and an increase in K14-p.Arg125Cys-positive aggregates in cells. Overexpression of the chaperone-associated ubiquitin ligase CHIP/STUB1 strongly reduced keratin aggregates through increased degradation of mutant K14. Using CHIP-p.Met1_Ala142del (DeltaTPR-CHIP), we demonstrated the involvement of Hsc70 and Hsp70 in mutant keratin degradation. Our data uncover common principles between EBS and other protein misfolding disorders, revealing that aggregation-prone keratins are targeted by components of the chaperone machinery. Thus, modulation of the chaperone machinery using small molecules may represent a novel therapeutic strategy for dominant EBS, allowing reformation of an intact keratin cytoskeleton.
Assuntos
Queratinas/metabolismo , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Epidermólise Bolhosa Simples/metabolismo , Epidermólise Bolhosa Simples/patologia , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Queratinas/química , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Estrutura Quaternária de Proteína , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Molecular chaperones are well known as facilitators of protein folding and assembly. However, in recent years multiple chaperone-assisted degradation pathways have also emerged, including CAP (chaperone-assisted proteasomal degradation), CASA (chaperone-assisted selective autophagy), and CMA (chaperone-mediated autophagy). Within these pathways chaperones facilitate the sorting of non-native proteins to the proteasome and the lysosomal compartment for disposal. Impairment of these pathways contributes to the development of cancer, myopathies, and neurodegenerative diseases. Chaperone-assisted degradation thus represents an essential aspect of cellular proteostasis, and its pharmacological modulation holds the promise to ameliorate some of the most devastating diseases of our time. Here, we discuss recent insights into molecular mechanisms underlying chaperone-assisted degradation in mammalian cells and highlight its biomedical relevance.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas/metabolismo , Animais , Autofagia/fisiologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.
Assuntos
Ligases/metabolismo , Chaperonas Moleculares/metabolismo , Cisteína Endopeptidases/metabolismo , Ligases/química , Substâncias Macromoleculares , Modelos Biológicos , Complexos Multienzimáticos/metabolismo , Família Multigênica , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
A major cellular catabolic pathway in neurons is macroautophagy/autophagy, through which misfolded or aggregation-prone proteins are sequestered into autophagosomes that fuse with lysosomes, and are degraded. MAPT (microtubule-associated protein tau) is one of the protein clients of autophagy. Given that accumulation of hyperphosphorylated MAPT contributes to the pathogenesis of Alzheimer disease and other tauopathies, decreasing endogenous MAPT levels has been shown to be beneficial to neuronal health in models of these diseases. A previous study demonstrated that the HSPA/HSP70 co-chaperone BAG3 (BCL2-associated athanogene 3) facilitates endogenous MAPT clearance through autophagy. These findings prompted us to further investigate the mechanisms underlying BAG3-mediated autophagy in the degradation of endogenous MAPT. Here we demonstrate for the first time that BAG3 plays an important role in autophagic flux in the neurites of mature neurons (20-24 days in vitro [DIV]) through interaction with the post-synaptic cytoskeleton protein SYNPO (synaptopodin). Loss of either BAG3 or SYNPO impeded the fusion of autophagosomes and lysosomes predominantly in the post-synaptic compartment. A block of autophagy leads to accumulation of the autophagic receptor protein SQSTM1/p62 (sequestosome 1) as well as MAPT phosphorylated at Ser262 (p-Ser262). Furthermore, p-Ser262 appears to accumulate in autophagosomes at post-synaptic densities. Overall these data provide evidence of a novel role for the co-chaperone BAG3 in synapses. In cooperation with SYNPO, it functions as part of a surveillance complex that facilitates the autophagic clearance of MAPT p-Ser262, and possibly other MAPT species at the post-synapse. This appears to be crucial for the maintenance of a healthy, functional synapse.Abbreviations: aa: amino acids; ACTB: actin beta; BafA1: bafilomycin A1; BAG3: BCL2 associated athanogene 3; CQ chloroquine; CTSL: cathepsin L; DIV: days in vitro; DLG4/PSD95: discs large MAGUK scaffold protein 4; HSPA/HSP70: heat shock protein family A (Hsp70); MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPT: microtubule associated protein tau; p-Ser262: MAPT phosphorylated at serine 262; p-Ser396/404: MAPT phosphorylated at serines 396 and 404; p-Thr231: MAPT phosphorylated at threonine 231; PBS: phosphate buffered saline; PK: proteinase K; scr: scrambled; shRNA: short hairpin RNA; SQSTM1/p62 sequestosome 1; SYN1: synapsin I; SYNPO synaptopodin; SYNPO2/myopodin: synaptopodin 2; VPS: vacuolar protein sorting.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas dos Microfilamentos/genética , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação , Densidade Pós-Sináptica/metabolismo , Proteólise , Ratos , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Sinapses/metabolismo , Domínios WW , Proteínas tau/químicaRESUMO
Chaperone-assisted selective autophagy (CASA) initiated by the cochaperone Bcl2-associated athanogene 3 (BAG3) represents an important mechanism for the disposal of misfolded and damaged proteins in mammalian cells. Under mechanical stress, the cochaperone cooperates with the small heat shock protein HSPB8 and the cytoskeleton-associated protein SYNPO2 to degrade force-unfolded forms of the actin-crosslinking protein filamin. This is essential for muscle maintenance in flies, fish, mice and men. Here, we identify the serine/threonine protein kinase 38 (STK38), which is part of the Hippo signaling network, as a novel interactor of BAG3. STK38 was previously shown to facilitate cytoskeleton assembly and to promote mitophagy as well as starvation and detachment induced autophagy. Significantly, our study reveals that STK38 exerts an inhibitory activity on BAG3-mediated autophagy. Inhibition relies on a disruption of the functional interplay of BAG3 with HSPB8 and SYNPO2 upon binding of STK38 to the cochaperone. Of note, STK38 attenuates CASA independently of its kinase activity, whereas previously established regulatory functions of STK38 involve target phosphorylation. The ability to exert different modes of regulation on central protein homeostasis (proteostasis) machineries apparently allows STK38 to coordinate the execution of diverse macroautophagy pathways and to balance cytoskeleton assembly and degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteostase/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Citoesqueleto/metabolismo , Filaminas/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas dos Microfilamentos , Mitofagia , Chaperonas Moleculares/metabolismo , Ligação Proteica , Proteômica , Transdução de Sinais , Estresse MecânicoRESUMO
Protein degradation in eukaryotic cells usually involves the attachment of a ubiquitin chain to a substrate protein and its subsequent sorting to the proteasome. Molecular mechanisms underlying the sorting process only recently began to emerge and rely on a cooperation of chaperone machineries and ubiquitin-chain recognition factors [1-3]. Here, we identify isoforms of the cochaperone HSJ1 as neuronal shuttling factors for ubiquitylated proteins. HSJ1 combines a J-domain that stimulates substrate loading onto the Hsc70 chaperone with ubiquitin interaction motifs (UIMs) involved in binding ubiquitylated chaperone clients. HSJ1 prevents client aggregation, shields clients against chain trimming by ubiquitin hydrolases, and stimulates their sorting to the proteasome. In this way, HSJ1 isoforms participate in ER-associated degradation (ERAD) and protect neurons against cytotoxic protein aggregation.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico/genética , Humanos , Imunoprecipitação , Luciferases , Modelos Biológicos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Peptídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismoRESUMO
Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.