RESUMO
Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the ß-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-Å resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/genética , Glaucoma/genética , Glicoproteínas/genética , Mutação , Proteínas do Citoesqueleto/química , Proteínas da Matriz Extracelular/química , Proteínas do Olho/química , Variação Genética/genética , Glicoproteínas/química , Células HEK293 , Humanos , Simulação de Dinâmica MolecularRESUMO
The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.
Assuntos
Glaucoma de Ângulo Aberto/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.
Assuntos
Amiloide/metabolismo , Ciclofilinas/metabolismo , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Amiloide/genética , Amiloide/ultraestrutura , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Transtornos Cognitivos/genética , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/fisiopatologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclosporina/farmacologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Doenças Neurodegenerativas/genética , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Tauopatias/genética , Tauopatias/metabolismo , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMO
Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation in vitro, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit in vitro tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and ex vivo slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation in vitro, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds.
Assuntos
Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas tau/antagonistas & inibidores , Células HEK293 , Humanos , Técnicas In VitroRESUMO
Gain-of-function mutations in the olfactomedin domain of the MYOC gene facilitate the toxic accumulation of amyloid-containing myocilin aggregates, hastening the onset of the prevalent ocular disorder primary open-angle glaucoma. Aggregation of wild-type myocilin has been reported in other glaucoma subtypes, suggesting broader relevance of misfolded myocilin across the disease spectrum, but the absence of myocilin does not cause disease. Thus, strategies aimed at eliminating myocilin could be therapeutically relevant for glaucoma. Here, a novel and selective Grp94 inhibitor reduced the levels of several mutant myocilin proteins as well as wild-type myocilin when forced to misfold in cells. This inhibitor rescued mutant myocilin toxicity in primary human trabecular meshwork cells. Mechanistically, in vitro kinetics studies demonstrate that Grp94 recognizes on-pathway aggregates of the myocilin olfactomedin domain (myoc-OLF), accelerates rates of aggregation and co-precipitates with myoc-OLF. These results indicate that aberrant myocilin quaternary structure drives Grp94 recognition, rather than peptide motifs exposed by unfolded protein. Inhibition of Grp94 ameliorates the effects of Grp94-accelerated myoc-OLF aggregation, and Grp94 remains in solution. In cells, when wild-type myocilin is driven to misfold and aggregate, it becomes a client of Grp94 and sensitive to Grp94 inhibition. Taken together, the interaction of Grp94 with myocilin aggregates can be manipulated by cellular environment and genetics; this process can be exploited with Grp94 inhibitors to promote the clearance of toxic forms of myocilin.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/metabolismo , Imidazóis/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Relação Dose-Resposta a Droga , Proteínas do Olho/genética , Regulação da Expressão Gênica , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/patologia , Glicoproteínas/genética , Células HEK293 , Humanos , Imidazóis/síntese química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Malha TrabecularRESUMO
BACKGROUND: Mutant myocilin accumulates in the endoplasmic reticulum for unknown reasons. RESULTS: Glucose-regulated protein (Grp) 94 depletion reduces mutant myocilin by engaging autophagy. CONCLUSION: Grp94 triages mutant myocilin through ER-associated degradation, subverting autophagy. SIGNIFICANCE: Treating glaucoma could be possible by inhibiting Grp94 and reducing its novel client, mutant myocilin. Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that ß-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.
Assuntos
Autofagia , Proteínas do Citoesqueleto/genética , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Mutação , Ligação ProteicaRESUMO
Tau aggregation and amyloidogenesis are common hallmarks for neurodegenerative disorders called tauopathies. The molecular chaperone network constitutes the cellular defense against insults such as tau aggregation. However, chaperone effects on tau are dichotomous. Loss of tau's microtubule-binding activity facilitates an inappropriate chaperone interaction that promotes an amyloidogenic tau conformation. Conversely, other chaperones are capable of promoting tau clearance. Here, we demonstrate that a critical contributor to tau triage is the DnaJ-binding domain of Hsp70 proteins. In particular, over-expression of the constitutive DnaJ, DnaJA1, mediated tau clearance, while knockdown facilitated tau accumulation. This clearance was not specific to distinct pathogenic tau species. The activity of DnaJA1 was attenuated by concomitant increases in Hsp70. Tau reductions facilitated by DnaJA1 were dependent on the integrity of lysines known to be poly-ubiquitinated in human Alzheimer's brain. In vivo, DnaJA1 and tau levels were inversely correlated. The effects of DnaJA1 were partially specific: DnaJA1 reduced the levels of a polyQ protein but had no significant effect on α-synuclein levels. These data suggest that DnaJA1 triages all tau species for ubiquitin-dependent clearance mechanisms. Moreover, the levels of DnaJA1 and Hsp70 seem to play against each other with regard to tau: as DnaJA1 levels increase, tau levels are reduced, but this can be prevented if Hsp70 levels are simultaneously induced. Thus, the DnaJ repertoire possibly represents a powerful set of genetic modifiers for tau pathogenesis. Further investigations could provide new insights about triage decisions that facilitate or prevent amyloidogenesis of tau and other proteins associated with neurodegenerative disease.