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1.
Mol Ther ; 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33545358

RESUMO

Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

2.
Acta Neuropathol Commun ; 9(1): 21, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33541434

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that affects motoneurons. Mutations in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpressing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfide isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfide bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defined. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1G93A/ERp57WT animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1G93A littermates at early-symptomatic stage, along with improved motor performance without affecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective effects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskeleton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifier at early stages by maintaining motoneuron connectivity.

3.
Proc Natl Acad Sci U S A ; 118(3)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33441483

RESUMO

Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

4.
ACS Infect Dis ; 6(12): 3174-3189, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33263384

RESUMO

Human coronaviruses (hCoVs) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite a high sequence similarity between SARS-CoV-1 and -2, each strain has a distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV nonstructural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43-an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologues from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response associated proteins, and antiviral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondria-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed antiviral therapeutics.

5.
Exp Cell Res ; : 112417, 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33301765

RESUMO

The endoplasmic reticulum (ER), responsible for processing approximately one-third of the human proteome including most secreted and membrane proteins, plays a pivotal role in protein homeostasis (proteostasis). Dysregulation of ER proteostasis has been implicated in a number of disease states. As such, continued efforts are directed at elucidating mechanisms of ER protein quality control which are mediated by transient and dynamic protein-protein interactions with molecular chaperones, co-chaperones, protein folding and trafficking factors that take place in and around the ER. Technological advances in mass spectrometry have played a pivotal role in characterizing and understanding these protein-protein interactions that dictate protein quality control mechanisms. Here, we highlight the recent progress from mass spectrometry-based investigation of ER protein quality control in revealing the topological arrangement of the proteostasis network, stress response mechanisms that adjust the ER proteostasis capacity, and disease specific changes in proteostasis network engagement. We close by providing a brief outlook on underexplored areas of ER proteostasis where mass spectrometry is a tool uniquely primed to further expand our understanding of the regulation and coordination of protein quality control processes in diverse diseases.

6.
Mol Cell Proteomics ; 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208410

RESUMO

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for ER-associated degradation (ERAD). Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

7.
Int J Mol Sci ; 21(16)2020 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-32796510

RESUMO

Autoimmune diabetes is a complex multifactorial disease with genetic and environmental factors playing pivotal roles. While many genes associated with the risk of diabetes have been identified to date, the mechanisms by which external triggers contribute to the genetic predisposition remain unclear. Here, we derived embryonic stem (ES) cell lines from diabetes-prone non-obese diabetic (NOD) and healthy C57BL/6 (B6) mice. While overall pluripotency markers were indistinguishable between newly derived NOD and B6 ES cells, we discovered several differentially expressed genes that normally are not expressed in ES cells. Several genes that reside in previously identified insulin-dependent diabetics (Idd) genomic regions were up-regulated in NOD ES cells. Gene set enrichment analysis showed that different groups of genes associated with immune functions are differentially expressed in NOD. Transcriptomic analysis of NOD blastocysts validated several differentially overexpressed Idd genes compared to B6. Genome-wide mapping of active histone modifications using ChIP-Seq supports active expression as the promoters and enhancers of activated genes are also marked by active histone modifications. We have also found that NOD ES cells secrete more inflammatory cytokines. Our data suggest that the known genetic predisposition of NOD to autoimmune diabetes leads to epigenetic instability of several Idd regions.

8.
Acta Neuropathol ; 140(5): 737-764, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32642868

RESUMO

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.

9.
bioRxiv ; 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32699849

RESUMO

Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 - an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca 2+ signaling and mitochondria biogenesis. We also identifiy nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N -linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

10.
Nat Chem Biol ; 16(10): 1052-1061, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690944

RESUMO

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

11.
Mol Cell Proteomics ; 20: 100008, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33581410

RESUMO

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for triiodothyronine and thyroxine hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism. Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several congenital hypothyroidism variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for endoplasmic reticulum-associated degradation. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for 1 Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

12.
Elife ; 82019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31149896

RESUMO

The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.


Assuntos
Fator 6 Ativador da Transcrição/antagonistas & inibidores , Organelas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Resposta a Proteínas não Dobradas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Organelas/efeitos dos fármacos , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Fenótipo , Ligação Proteica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
13.
Cell Chem Biol ; 26(7): 913-925.e4, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31105062

RESUMO

Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. We employ a quantitative interactomics platform to define how ATF6 activation reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with light-chain amyloidosis (AL). Using this platform, we show that ATF6 activation increases the targeting of this destabilized LC to a subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion. Our results define a molecular basis for the ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteínas Amiloidogênicas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Fator 6 Ativador da Transcrição/imunologia , Proteínas Amiloidogênicas/fisiologia , Amiloidose/imunologia , Amiloidose/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células HEK293 , Humanos , Chaperonas Moleculares , Proteômica/métodos , Fatores de Transcrição/metabolismo
14.
ACS Chem Biol ; 14(4): 784-795, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30821953

RESUMO

Cellular proteostasis is maintained by stress-responsive signaling pathways such as the heat shock response (HSR), the oxidative stress response (OSR), and the unfolded protein response (UPR). Activation of these pathways results in the transcriptional upregulation of select subsets of stress-responsive genes that restore proteostasis and adapt cellular physiology to promote recovery following various types of acute insult. The capacity for these pathways to regulate cellular proteostasis makes them attractive therapeutic targets for correcting proteostasis defects associated with diverse diseases. High-throughput screening (HTS) using cell-based reporter assays is highly effective for identifying putative activators of stress-responsive signaling pathways. However, the development of these compounds is hampered by the lack of medium-throughput assays to define compound potency and selectivity for a given pathway. Here, we describe a targeted RNA sequencing (RNAseq) assay that allows cost-effective, medium-throughput screening of stress-responsive signaling pathway activation. We demonstrate that this assay allows deconvolution of stress-responsive signaling activated by chemical genetic or pharmacologic agents. Furthermore, we use this assay to define the selectivity of putative OSR and HSR activating compounds previously identified by HTS. Our results demonstrate the potential for integrating this adaptable targeted RNAseq assay into screening programs focused on developing pharmacologic activators of stress-responsive signaling pathways.


Assuntos
Fatores de Transcrição de Choque Térmico/metabolismo , Fator de Transcrição NF-E2/metabolismo , Estresse Oxidativo , Proteostase , Análise de Sequência de RNA , Resposta a Proteínas não Dobradas , Animais , Células HEK293 , Humanos , Camundongos , Proteostase/fisiologia , Transdução de Sinais
15.
Nat Commun ; 10(1): 187, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643122

RESUMO

Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo. Here we show, using a mouse model of myocardial ischemia/reperfusion, that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 is also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Infarto Cerebral/prevenção & controle , Nefropatias/prevenção & controle , Infarto do Miocárdio/prevenção & controle , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Fator 6 Ativador da Transcrição/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Infarto Cerebral/etiologia , Infarto Cerebral/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Ventrículos do Coração/patologia , Humanos , Rim/irrigação sanguínea , Rim/patologia , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos , Cultura Primária de Células , Substâncias Protetoras/uso terapêutico , Proteostase/efeitos dos fármacos , Ratos , Traumatismo por Reperfusão/etiologia , Resultado do Tratamento , Resposta a Proteínas não Dobradas/efeitos dos fármacos
16.
Elife ; 72018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30084354

RESUMO

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (147) was previously identified (Plate et al., 2016) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that 147-dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by 147 include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs 147-dependent induction of the ATF6-target gene, BiP, implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with 147. Thus, 147 is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.


Assuntos
Fator 6 Ativador da Transcrição/genética , Amidas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fenilpropionatos/farmacologia , Pró-Fármacos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética
17.
Sci Signal ; 11(517)2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29440509

RESUMO

ATF6 encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6ß results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with ATF6 mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.


Assuntos
Fator 6 Ativador da Transcrição/genética , Diferenciação Celular/genética , Mesoderma/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 6 Ativador da Transcrição/agonistas , Fator 6 Ativador da Transcrição/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesoderma/citologia , Mutação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
18.
J Am Chem Soc ; 140(1): 200-210, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29265822

RESUMO

Drug candidates are generally discovered using biochemical screens employing an isolated target protein or by utilizing cell-based phenotypic assays. Both noncovalent and covalent hits emerge from such endeavors. Herein, we exemplify an "Inverse Drug Discovery" strategy in which organic compounds of intermediate complexity harboring weak, but activatable, electrophiles are matched with the protein(s) they react with in cells or cell lysate. An alkyne substructure in each candidate small molecule enables affinity chromatography-mass spectrometry, which produces a list of proteins that each distinct compound reacts with. A notable feature of this approach is that it is agnostic with respect to the cellular proteins targeted. To illustrate this strategy, we employed aryl fluorosulfates, an underexplored class of sulfur(VI) halides, that are generally unreactive unless activated by protein binding. Reversible aryl fluorosulfate binding, correct juxtaposition of protein side chain functional groups, and transition-state stabilization of the S(VI) exchange reaction all seem to be critical for conjugate formation. The aryl fluorosulfates studied thus far exhibit chemoselective reactivity toward Lys and, particularly, Tyr side chains, and can be used to target nonenzymes (e.g., a hormone carrier or a small-molecule carrier protein) as well as enzymes. The "Inverse Drug Discovery" strategy should be particularly attractive as a means to explore latent electrophiles not typically used in medicinal chemistry efforts, until one reacts with a protein target of exceptional interest. Structure-activity data can then be used to enhance the selectivity of conjugate formation or the covalent probe can be used as a competitor to develop noncovalent drug candidates. Here we use the "Inverse Drug Discovery" platform to identify and validate covalent ligands for 11 different human proteins. In the case of one of these proteins, we have identified and validated a small-molecule probe for the first time.


Assuntos
Descoberta de Drogas , Proteínas/análise , Ésteres do Ácido Sulfúrico/química , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular
19.
Sci Transl Med ; 9(407)2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28904227

RESUMO

Increasing evidence supports the hypothesis that soluble misfolded protein assemblies contribute to the degeneration of postmitotic tissue in amyloid diseases. However, there is a dearth of reliable nonantibody-based probes for selectively detecting oligomeric aggregate structures circulating in plasma or deposited in tissues, making it difficult to scrutinize this hypothesis in patients. Hence, understanding the structure-proteotoxicity relationships driving amyloid diseases remains challenging, hampering the development of early diagnostic and novel treatment strategies. We report peptide-based probes that selectively label misfolded transthyretin (TTR) oligomers circulating in the plasma of TTR hereditary amyloidosis patients exhibiting a predominant neuropathic phenotype. These probes revealed that there are much fewer misfolded TTR oligomers in healthy controls, in asymptomatic carriers of mutations linked to amyloid polyneuropathy, and in patients with TTR-associated cardiomyopathies. The absence of misfolded TTR oligomers in the plasma of cardiomyopathy patients suggests that the tissue tropism observed in the TTR amyloidoses is structure-based. Misfolded oligomers decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (tafamidis or liver transplant-mediated gene therapy). In a subset of TTR amyloid polyneuropathy patients, the probes also detected a circulating TTR fragment that disappeared after tafamidis treatment. Proteomic analysis of the isolated TTR oligomers revealed a specific patient-associated signature composed of proteins that likely associate with the circulating TTR oligomers. Quantification of plasma oligomer concentrations using peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.


Assuntos
Amiloidose Familiar/sangue , Sondas Moleculares/química , Peptídeos/química , Pré-Albumina/metabolismo , Dobramento de Proteína , Multimerização Proteica , Amiloidose Familiar/genética , Benzoxazóis/farmacologia , Estudos de Casos e Controles , Reagentes para Ligações Cruzadas/química , Diazometano/química , Genótipo , Humanos , Íons , Luz , Peso Molecular , Pré-Albumina/química , Estrutura Secundária de Proteína , Proteólise , Proteômica , Solubilidade
20.
EMBO J ; 36(15): 2296-2309, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28655754

RESUMO

ERdj3/DNAJB11 is an endoplasmic reticulum (ER)-targeted HSP40 co-chaperone that performs multifaceted functions involved in coordinating ER and extracellular proteostasis. Here, we show that ERdj3 assembles into a native tetramer that is distinct from the dimeric structure observed for other HSP40 co-chaperones. An electron microscopy structural model of full-length ERdj3 shows that these tetramers are arranged as a dimer of dimers formed by distinct inter-subunit interactions involving ERdj3 domain II and domain III Targeted deletion of residues 175-190 within domain II renders ERdj3 a stable dimer that is folded and efficiently secreted from mammalian cells. This dimeric ERdj3 shows impaired substrate binding both in the ER and extracellular environments and reduced interactions with the ER HSP70 chaperone BiP. Furthermore, we show that overexpression of dimeric ERdj3 exacerbates ER stress-dependent reductions in the secretion of a destabilized, aggregation-prone protein and increases its accumulation as soluble oligomers in extracellular environments. These results reveal ERdj3 tetramerization as an important structural framework for ERdj3 functions involved in coordinating ER and extracellular proteostasis in the presence and absence of ER stress.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Multimerização Proteica , Linhagem Celular , Células Epiteliais/fisiologia , Proteínas de Choque Térmico HSP40/ultraestrutura , Humanos , Microscopia Eletrônica , Mapeamento de Interação de Proteínas
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