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1.
Biochem J ; 478(24): 4203-4220, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34821356

RESUMEN

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis. To this end, we developed a novel yeast expression system for SLC26A9. This facile system has been used extensively with other ion channels and transporters to screen for factors that oversee protein folding checkpoints. As commonly observed for other channels and transporters, we first noted that a substantial fraction of SLC26A9 is targeted for endoplasmic reticulum associated degradation (ERAD), which destroys folding-compromised proteins in the early secretory pathway. We next discovered that ERAD selection requires the Hsp70 chaperone, which can play a vital role in ERAD substrate selection. We then created SLC26A9 mutants and found that the transmembrane-rich domain of SLC26A9 was quite stable, whereas the soluble cytosolic STAS domain was responsible for Hsp70-dependent ERAD. To support data obtained in the yeast model, we were able to recapitulate Hsp70-facilitated ERAD of the STAS domain in human tissue culture cells. These results indicate that a critical barrier to nascent membrane protein folding can reside within a specific soluble domain, one that is monitored by components associated with the ERAD machinery.


Asunto(s)
Antiportadores/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/metabolismo , Modelos Biológicos , Transportadores de Sulfato/metabolismo , Antiportadores/genética , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Humanos , Dominios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transportadores de Sulfato/genética
2.
J Biol Chem ; 293(28): 11006-11021, 2018 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-29784874

RESUMEN

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.


Asunto(s)
Arrestina/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Modelos Biológicos , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arrestina/genética , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética
3.
Bioorg Med Chem ; 27(1): 79-91, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30528127

RESUMEN

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson's Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100 µm, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Activadores de Enzimas/farmacología , Ésteres/farmacología , Proteínas HSP70 de Choque Térmico/agonistas , Multimerización de Proteína/efectos de los fármacos , Pirimidinonas/farmacología , Línea Celular Tumoral , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activadores de Enzimas/síntesis química , Activadores de Enzimas/química , Activadores de Enzimas/toxicidad , Ésteres/síntesis química , Ésteres/química , Ésteres/toxicidad , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Estructura Molecular , Pliegue de Proteína/efectos de los fármacos , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/toxicidad , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , alfa-Sinucleína/agonistas , alfa-Sinucleína/metabolismo
4.
Bioorg Med Chem Lett ; 26(20): 5087-5091, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27624078

RESUMEN

Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication. To improve the antiviral activity of the existing class of compounds, we performed Biginelli and modified multi-component reactions to obtain new 3,4-dihydropyrimidin-2(1H)-ones and -thiones for biological evaluation. We also compared how substituents at the N-1 versus N-3 position in the pyrimidine affect activity. We discovered that AMT580-043, a N-3 alkylated dihydropyrimidin-2(1H)-thione, inhibits the replication of a disease-causing polyomavirus in cell culture more potently than an existing drug, cidofovir.


Asunto(s)
Antivirales/farmacología , Poliomavirus/efectos de los fármacos , Pirimidinonas/farmacología , Animales , Antivirales/química , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Pirimidinonas/química , Relación Estructura-Actividad
5.
Physiol Genomics ; 47(6): 198-214, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25759377

RESUMEN

Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses.


Asunto(s)
Proteínas de la Membrana/genética , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Canales Epiteliales de Sodio , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Ontología de Genes , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Pliegue de Proteína , Regulón/genética , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/genética , Regulación hacia Arriba/genética
6.
J Biol Chem ; 289(45): 31591-604, 2014 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-25258318

RESUMEN

Ypk1, the yeast homolog of the human serum- and glucocorticoid-induced kinase (Sgk1), affects diverse cellular activities, including sphingolipid homeostasis. We now report that Ypk1 also impacts the turnover of the major phospholipid, phosphatidylcholine (PC). Pulse-chase radiolabeling reveals that a ypk1Δ mutant exhibits increased PC deacylation and glycerophosphocholine production compared with wild type yeast. Deletion of PLB1, a gene encoding a B-type phospholipase that hydrolyzes PC, in a ypk1Δ mutant curtails the increased PC deacylation. In contrast to previous data, we find that Plb1 resides in the ER and in the medium. Consistent with a link between Ypk1 and Plb1, the levels of both Plb1 protein and PLB1 message are elevated in a ypk1Δ strain compared with wild type yeast. Furthermore, deletion of PLB1 in a ypk1Δ mutant exacerbates phenotypes associated with loss of YPK1, including slowed growth and sensitivity to cell wall perturbation, suggesting that increased Plb1 activity buffers against the loss of Ypk1. Because Plb1 lacks a consensus phosphorylation site for Ypk1, we probed other processes under the control of Ypk1 that might be linked to PC turnover. Inhibition of sphingolipid biosynthesis by the drug myriocin or through utilization of a lcb1-100 mutant results in increased PLB1 expression. Furthermore, we discovered that the increase in PLB1 expression observed upon inhibition of sphingolipid synthesis or loss of Ypk1 is under the control of the Crz1 transcription factor. Taken together, these results suggest a functional interaction between Ypk1 and Plb1 in which altered sphingolipid metabolism up-regulates PLB1 expression via Crz1.


Asunto(s)
Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Lisofosfolipasa/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Alelos , Colina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos Monoinsaturados/química , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glicerilfosforilcolina/metabolismo , Homeostasis , Hidrólisis , Lípidos/química , Proteínas de la Membrana/metabolismo , Mutación , Fenotipo , Fosforilación , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Factores de Transcripción/metabolismo
7.
J Biol Chem ; 288(18): 13124-35, 2013 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-23482560

RESUMEN

The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Complejos Multiproteicos/metabolismo , Proteolisis , Receptores de Droga/metabolismo , Simportadores/metabolismo , Animales , Línea Celular , Perros , Retículo Endoplásmico/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Ratones , Complejos Multiproteicos/genética , Pliegue de Proteína , Receptores de Droga/genética , Miembro 3 de la Familia de Transportadores de Soluto 12 , Simportadores/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
8.
Biochim Biophys Acta ; 1833(11): 2447-57, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23557783

RESUMEN

All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation. What was less clear was how secreted and integral membrane proteins, which in eukaryotes enter the endoplasmic reticulum (ER), were subject to quality control decisions. In this review, we highlight early studies that ultimately led to the discovery that secreted and integral membrane proteins also utilize several components that constitute the cytoplasmic quality control machinery. This component of the cellular quality control pathway is known as ER associated degradation, or ERAD. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Humanos , Proteolisis
9.
J Biol Chem ; 286(51): 43611-43621, 2011 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-22027832

RESUMEN

The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) mediates salt reabsorption in the distal nephron of the kidney and is the target of thiazide diuretics, which are commonly prescribed to treat hypertension. Mutations in NCC also give rise to Gitelman syndrome, a hereditary salt-wasting disorder thought in most cases to arise from impaired NCC biogenesis through enhanced endoplasmic reticulum-associated degradation (ERAD). Because the machinery that mediates NCC quality control is completely undefined, we employed yeast as a model heterologous expression system to identify factors involved in NCC degradation. We confirmed that NCC was a bona fide ERAD substrate in yeast, as the majority of NCC polypeptide was integrated into ER membranes, and its turnover rate was sensitive to proteasome inhibition. NCC degradation was primarily dependent on the ER membrane-associated E3 ubiquitin ligase Hrd1. Whereas several ER luminal chaperones were dispensable for NCC ERAD, NCC ubiquitination and degradation required the activity of Ssa1, a cytoplasmic Hsp70 chaperone. Compatible findings were observed when NCC was expressed in mammalian kidney cells, as the cotransporter was polyubiquitinated and degraded by the proteasome, and mammalian cytoplasmic Hsp70 (Hsp72) coexpression stimulated the degradation of newly synthesized NCC. Hsp70 also preferentially associated with the ER-localized NCC glycosylated species, indicating that cytoplasmic Hsp70 plays a critical role in selecting immature forms of NCC for ERAD. Together, these results provide the first survey of components involved in the ERAD of a mammalian SLC12 cation chloride cotransporter and provide a framework for future studies on NCC ER quality control.


Asunto(s)
Retículo Endoplásmico/metabolismo , Cloruro de Sodio/química , Tiazidas/química , Animales , Transporte Biológico , Cationes , Línea Celular , Cloruros/química , Cicloheximida/farmacología , Perros , Glicosilación , Células HEK293 , Proteínas HSP70 de Choque Térmico/química , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Saccharomyces cerevisiae/metabolismo , Ubiquitina/química
10.
Neurotherapeutics ; 18(4): 2541-2564, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34528172

RESUMEN

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein+ inclusions more robustly in male-derived neurons. Because Hsp70 is released into extracellular compartments and may restrict cell-to-cell transmission/amplification of α-synucleinopathy, we then tested the effects of low-endotoxin, exogenous Hsp70 (eHsp70) in primary hippocampal cultures. eHsp70 was taken up by and reduced α-synuclein+ inclusions in cells of both sexes, but pharmacological suppression of Hsp70 function attenuated the inhibitory effect of eHsp70 on perinuclear inclusions only in male neurons. In 20-month-old male mice infused with α-synuclein fibrils in the olfactory bulb, daily intranasal eHsp70 delivery also reduced inclusion numbers and the time to locate buried food. eHsp70 penetrated the limbic system and spinal cord of male mice within 3 h but was cleared within 72 h. Unexpectedly, no evidence of eHsp70 uptake from nose into brain was observed in females. A trend towards higher expression of inducible Hsp70-but not constitutive Hsp70 or Hsp40-was observed in amygdala tissues from male subjects with Lewy body disorders compared to unaffected male controls, supporting the importance of this chaperone in human disease. Women expressed higher amygdalar Hsp70 levels compared to men, regardless of disease status. Together, these data provide a new link between biological sex and a key chaperone that orchestrates proteostasis.


Asunto(s)
Proteínas HSP70 de Choque Térmico , Enfermedad por Cuerpos de Lewy , Factores Sexuales , Sinucleinopatías , Animales , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Enfermedad por Cuerpos de Lewy/metabolismo , Masculino , Ratones , Bulbo Olfatorio , Ratas Sprague-Dawley , Sinucleinopatías/metabolismo , alfa-Sinucleína/metabolismo
11.
Artículo en Inglés | MEDLINE | ID: mdl-30670468

RESUMEN

Misfolded proteins compromise cellular homeostasis. This is especially problematic in the endoplasmic reticulum (ER), which is a high-capacity protein-folding compartment and whose function requires stringent protein quality-control systems. Multiprotein complexes in the ER are able to identify, remove, ubiquitinate, and deliver misfolded proteins to the 26S proteasome for degradation in the cytosol, and these events are collectively termed ER-associated degradation, or ERAD. Several steps in the ERAD pathway are facilitated by molecular chaperone networks, and the importance of ERAD is highlighted by the fact that this pathway is linked to numerous protein conformational diseases. In this review, we discuss the factors that constitute the ERAD machinery and detail how each step in the pathway occurs. We then highlight the underlying pathophysiology of protein conformational diseases associated with ERAD.


Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Deficiencias en la Proteostasis/patología , Citoplasma/metabolismo , Genoma Humano , Homeostasis , Humanos , Mutación , Péptidos/metabolismo , Polisacáridos/química , Pliegue de Proteína , Proteostasis , Control de Calidad
12.
Science ; 365(6448): 53-60, 2019 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-31273116

RESUMEN

The COPII-cargo adaptor complex Lst1-Sec23 selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that uses ER-resident receptors. Working in yeast, we found an unexpected role for Lst1-Sec23 in ER-phagy that was independent from its function in secretion. Up-regulation of the stress-inducible ER-phagy receptor Atg40 induced the association of Lst1-Sec23 with Atg40 at distinct ER domains to package ER into autophagosomes. Lst1-mediated ER-phagy played a vital role in maintaining cellular homeostasis by preventing the accumulation of an aggregation-prone protein in the ER. Lst1 function appears to be conserved because its mammalian homolog, SEC24C, was also required for ER-phagy.


Asunto(s)
Autofagia , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Estrés del Retículo Endoplásmico , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Respuesta de Proteína Desplegada
13.
Neuron ; 102(2): 321-338.e8, 2019 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-30826182

RESUMEN

TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Transición de Fase , ARN/metabolismo , Estrés Fisiológico , Proteinopatías TDP-43/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/metabolismo , Células HEK293 , Humanos , Cuerpos de Inclusión , Oligonucleótidos , Optogenética
14.
Arch Biochem Biophys ; 478(2): 167-74, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18706386

RESUMEN

We previously described many Hsp70 Ssa1p mutants that impair [PSI(+)] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1(L483W) ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones. Ssa1(A17V) and Ssa1(R34K) ATPase activities were nearly wild type but both showed increased stimulation by substrates. Peptide binding and reactivation of denatured luciferase were enhanced in Ssa1(A17V) and Ssa1(R34K) but compromised in Ssa1(L483W). The nucleotide exchange factor Fes1 influenced ATPase of wild type Ssa1 and each mutant differently. Partial protease digestion uncovered similar and distinct conformational changes of the substrate-binding domain among the three mutants. Our data suggest that prion-impairing mutations of Ssa1 can increase or decrease substrate interactions, alter the Hsp70 reaction cycle at different points and impair normal NBD-SBD cooperation.


Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Priones/genética , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Sustitución de Aminoácidos , Sitios de Unión/genética , Genes Fúngicos , Proteínas HSP70 de Choque Térmico/química , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
15.
Sci Rep ; 7(1): 1789, 2017 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-28496195

RESUMEN

C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase. Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain. In contrast to nearly all homodimeric proteins, CHIP is asymmetric. To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly. We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box. We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization. This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues. Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion. The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites. Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function. This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly.


Asunto(s)
Multimerización de Proteína , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Activación Enzimática , Humanos , Modelos Moleculares , Complejos Multiproteicos , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas
16.
J Mol Biol ; 427(18): 2948-65, 2015 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-25913688

RESUMEN

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L, as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild-type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants' substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1-45 allele.


Asunto(s)
Adenosina Trifosfatasas/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/biosíntesis , Adenosina Trifosfatasas/química , Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/química , Respuesta al Choque Térmico , Mitocondrias/metabolismo , Chaperonas Moleculares/química , Mutación , Proteolisis , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Especificidad por Sustrato
17.
Mol Biol Cell ; 25(2): 276-89, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24227888

RESUMEN

Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum-associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.


Asunto(s)
Membrana Celular/metabolismo , Degradación Asociada con el Retículo Endoplásmico/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Canales de Potasio de Rectificación Interna/genética , Membrana Celular/genética , Endosomas/genética , Endosomas/metabolismo , Regulación de la Expresión Génica , Humanos , Canales de Potasio de Rectificación Interna/metabolismo , Transporte de Proteínas , Propiedades de Superficie
18.
Cell Stress Chaperones ; 16(4): 389-401, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21191678

RESUMEN

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70-Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70-PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70-Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.


Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Adenosina Trifosfatasas/metabolismo , Citosol/metabolismo , Expresión Génica , Hidrólisis , Plasmodium falciparum/genética , Regulación hacia Arriba
19.
Virology ; 357(1): 102-13, 2007 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-16959286

RESUMEN

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner. Rep-NPM interactions were verified by co-immunofluorescence and chemical cross-linking studies. We have found that there is demonstrable, but limited co-localization between Rep and NPM in co-infected cells. In contrast, there was significant co-localization between NPM and AAV Cap proteins. In vitro experiments using purified MBPRep78 and NPM show that NPM stimulates MBPRep78 interactions with the AAV ITR as well as endonuclease activity. These studies suggest that NPM plays a role in AAV amplification affecting Rep function and virion assembly.


Asunto(s)
Nucléolo Celular/metabolismo , Dependovirus/fisiología , Proteínas Nucleares/fisiología , Virión/fisiología , Nucléolo Celular/virología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dependovirus/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Nucleofosmina , Unión Proteica , Proteínas Virales/genética , Proteínas Virales/metabolismo , Ensamble de Virus
20.
Yeast ; 23(16): 1151-66, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17133623

RESUMEN

The Mig1 DNA-binding protein of Saccharomyces cerevisiae was expressed and purified from yeast and the physical properties were characterized by several methods, including gel filtration, sucrose gradient sedimentation and native gel electrophoresis. Purified Mig1 exists as a monomer with a Stokes' radius of 48 A and a sedimentation coefficient of 3.55 S. Mig1 has an elongated shape with a frictional coefficient of 1.83. The K(d) of purified Mig1 for the SUC2 A site is 2.8 nM and for SUC2 B site 25.8 nM; these values were similar for Mig1 purified from repressed and derepressed cells. Full-length Mig1 expressed in yeast binds more tightly to SUC2 B than bacterially expressed GST-Mig1. Sucrose gradient sedimentation resolved a larger molecular weight form of Mig1 in whole-cell extracts that was not seen in purified samples and may represent a complex with another protein. This complex is found within the nucleus and is seen only in repressed cells. Mig1 exists in multiple phosphorylation states and only less phosphorylated forms of Mig1 are associated with this complex.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Sitios de Unión , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis/métodos , Regulación Fúngica de la Expresión Génica , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/aislamiento & purificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , beta-Fructofuranosidasa/genética
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