RESUMEN
This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.
Asunto(s)
Adenosina Trifosfato/química , Chaperoninas/química , Conformación Proteica , Pliegue de Proteína , Aminoácidos/química , Animales , Dióxido de Carbono/química , Citosol/metabolismo , Dimerización , Proteínas de Choque Térmico/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Ratones , Mitocondrias/metabolismo , Mutación , Neurospora/metabolismo , Desnaturalización Proteica , Ribonucleasa Pancreática/química , Ribulosa-Bifosfato Carboxilasa/química , Propiedades de Superficie , TemperaturaRESUMEN
Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.
Asunto(s)
Encéfalo/patología , Proteínas del Choque Térmico HSP110/metabolismo , Enfermedad de Parkinson/etiología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP110/genética , Humanos , Ratones Transgénicos , Enfermedad de Parkinson/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Sinucleinopatías/genética , Sinucleinopatías/mortalidad , Sinucleinopatías/patología , alfa-Sinucleína/genéticaRESUMEN
Extending from a study we recently published examining the antitrypanosomal effects of a series of GroEL/ES inhibitors based on a pseudosymmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria, EC50 values of the most potent analogs were in the 1-2 µM range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC50 values in the 1-10 µM range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC50 values > 20 µM).
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Antibacterianos/efectos adversos , Proteínas Bacterianas/antagonistas & inhibidores , Calorimetría/métodos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chaperonina 10/química , Chaperonina 10/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Sulfonamidas/química , Tiofenos/químicaRESUMEN
The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies.
Asunto(s)
Chaperonina 60/metabolismo , Pliegue de Proteína , Rhodospirillum rubrum/enzimología , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/metabolismo , Escherichia coli/enzimología , Modelos Moleculares , Unión Proteica , Dominios ProteicosRESUMEN
Basic cellular research is a trail. One follows one's nose toward what might be new understanding. When that leads to a need to employ unfamiliar or novel technology, it's both exciting and very worthwhile to form collaborations. Our early studies of chaperonins support such a philosophy, as detailed in the two stories that follow, written in deep appreciation of recognition by the E.B. Wilson Medal of the American Society for Cell Biology.
Asunto(s)
Chaperoninas/metabolismo , Colaboración Intersectorial , Investigación , Chaperoninas/genética , Humanos , Pliegue de Proteína , Proyectos de InvestigaciónRESUMEN
Recent studies have reported spread of pathogenic proteins in the mammalian nervous system, but whether nonpathogenic ones spread is unknown. We initially investigated whether spread of a mutant amyotrophic lateral sclerosis-associated cytosolic superoxide dismutase 1 (SOD1) protein between motor neurons could be detected in intact chimeric mice. Eight-cell embryos from G85R SOD1YFP and G85R SOD1CFP mice were aggregated, and spinal cords of adult chimeric progeny were examined for motor neurons with cytosolic double fluorescence. By 3 mo of age, we observed extensive double fluorescence, including in amyotrophic lateral sclerosis-affected cranial nerve motor nuclei but not in the relatively spared extraocular nuclei. Chimeras of nonpathogenic wtSOD1YFP and G85R SOD1CFP also exhibited double fluorescence. In a third chimera, mitochondrial mCherry did not transfer to G85R SOD1YFP motor neurons, suggesting that neither RNA nor organelles transfer, but mito-mCherry neurons received G85R SOD1YFP. In a chimera of ChAT promoter-EGFP and mito-mCherry, EGFP efficiently transferred to mito-mCherry+ cells. Thus, nonpathogenic cytosolic proteins appear capable of transfer. During study of both the SOD1FP and EGFP chimeras, we observed fluorescence also in small cells neighboring the motor neurons, identified as mature gray matter oligodendrocytes. Double fluorescence in the G85R SOD1FP chimera and observation of the temporal development of fluorescence first in motor neurons and then in these oligodendrocytes suggest that they may be mediators of transfer of cytosolic proteins between motor neurons.
Asunto(s)
Citosol/metabolismo , Neuronas Motoras/patología , Proteínas/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/fisiología , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Médula Espinal/metabolismoRESUMEN
Mutations that alter levels of Slack (KCNT1) Na+-activated K+ current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica, a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of â¼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na+ from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na+-activated K+ channels in neurons.SIGNIFICANCE STATEMENT Slack Na+-activated K+ channels (KCNT1, KNa1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal development and function. We find that injection of oligomers of mutant superoxide dismutase 1 (SOD1) into the cytoplasm of invertebrate neurons rapidly suppresses these Na+-activated K+ currents and that this effect is mediated by a MAP kinase cascade, including ASK1 and c-Jun N-terminal kinase. Because amyotrophic lateral sclerosis is a fatal adult-onset neurodegenerative disease produced by mutations in SOD1 that cause the enzyme to form toxic oligomers, our findings suggest that suppression of Slack channels may be an early step in the progression of the disease.
Asunto(s)
Potenciales de la Membrana/genética , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Canales de Potasio/metabolismo , Superóxido Dismutasa-1/genética , Animales , Aplysia/citología , Biofisica , Células Cultivadas , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Ganglios de Invertebrados/citología , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Microinyecciones , Morfolinos/farmacología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de potasio activados por Sodio , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sodio/farmacología , Superóxido Dismutasa-1/químicaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease prominently featuring motor neuron (MN) loss and paralysis. A recent study using whole-cell patch clamp recording of MNs in acute spinal cord slices from symptomatic adult ALS mice showed that the fastest firing MNs are preferentially lost. To measure the in vivo effects of such loss, awake symptomatic-stage ALS mice performing self-initiated walking on a wheel were studied. Both single-unit extracellular recordings within spinal cord MN pools for lower leg flexor and extensor muscles and the electromyograms (EMGs) of the corresponding muscles were recorded. In the ALS mice, we observed absent or truncated high-frequency firing of MNs at the appropriate time in the step cycle and step-to-step variability of the EMG, as well as flexor-extensor coactivation. In turn, kinematic analysis of walking showed step-to-step variability of gait. At the MN level, the higher frequencies absent from recordings from mutant mice corresponded with the upper range of frequencies observed for fast-firing MNs in earlier slice measurements. These results suggest that, in SOD1-linked ALS mice, symptoms are a product of abnormal MN firing due at least in part to loss of neurons that fire at high frequency, associated with altered EMG patterns and hindlimb kinematics during gait.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Marcha/fisiología , Neuronas Motoras/fisiología , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Electromiografía , Miembro Posterior/fisiopatología , Ratones , Ratones Transgénicos , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , MutaciónRESUMEN
Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis-HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1µM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.
Asunto(s)
Antiprotozoarios/farmacología , Chaperonina 10/efectos de los fármacos , Chaperonina 60/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-µM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.
Asunto(s)
Antibacterianos/farmacología , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Línea Celular , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Bacterias Gramnegativas/enzimología , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoter-driven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from â¼5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery.
Asunto(s)
Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas del Choque Térmico HSP110/metabolismo , Neuronas Motoras/metabolismo , Superóxido Dismutasa-1/metabolismo , Tasa de Supervivencia , Esclerosis Amiotrófica Lateral/genética , Animales , Femenino , Proteínas del Choque Térmico HSP110/genética , Masculino , Ratones , Ratones Transgénicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neuronas Motoras/patología , Pliegue de Proteína , Superóxido Dismutasa-1/genéticaRESUMEN
A recent hydrogen-deuterium exchange study of folding of the GroEL/GroES-dependent bacterial enzyme DapA has suggested that the DapA folding pathway when free in solution may differ from the folding pathway used in the presence of the GroEL/GroES chaperonin. Here, we have investigated whether DapA aggregation might be occurring in free solution under the conditions of the exchange experiment, as this would confound interpretation of the pathway predictions. Dynamic light scattering (DLS) data, sedimentation analysis and refolding yield indicate that significant aggregation occurs upon dilution of DapA from denaturant, bringing into question the earlier conclusion that different folding pathways occur in the absence and presence of the chaperonin system.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Proteínas de Escherichia coli/química , Hidroliasas/química , Agregado de Proteínas , Pliegue de Proteína , SolucionesRESUMEN
Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) affects motor neurons (MNs) in the brain and spinal cord. Understanding the pathophysiology of this condition seems crucial for therapeutic design, yet few electrophysiological studies in actively degenerating animal models have been reported. Here, we report a novel preparation of acute slices from adult mouse spinal cord, allowing visualized whole cell patch-clamp recordings of fluorescent lumbar MN cell bodies from ChAT-eGFP or superoxide dismutase 1-yellow fluorescent protein (SOD1YFP) transgenic animals up to 6 mo of age. We examined 11 intrinsic electrophysiologic properties of adult ChAT-eGFP mouse MNs and classified them into four subtypes based on these parameters. The subtypes could be principally correlated with instantaneous (initial) and steady-state firing rates. We used retrograde tracing using fluorescent dye injected into fast or slow twitch lower extremity muscle with slice recordings from the fluorescent-labeled lumbar MN cell bodies to establish that fast and slow firing MNs are connected with fast and slow twitch muscle, respectively. In a G85R SOD1YFP transgenic mouse model of ALS, which becomes paralyzed by 5-6 mo, where MN cell bodies are fluorescent, enabling the same type of recording from spinal cord tissue slices, we observed that all four MN subtypes were present at 2 mo of age. At 4 mo, by which time substantial neuronal SOD1YFP aggregation and cell loss has occurred and symptoms have developed, one of the fast firing subtypes that innvervates fast twitch muscle was lost. These results begin to describe an order of the pathophysiologic events in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Neuronas Motoras/fisiología , Médula Espinal/patología , Superóxido Dismutasa/fisiología , Esclerosis Amiotrófica Lateral/enzimología , Animales , Ratones , Neuronas Motoras/patología , Técnicas de Placa-Clamp , Superóxido Dismutasa-1RESUMEN
Lipofuscin, or aging pigment, is accreted as red autofluorescence in the lysosomes of motor neuron cell bodies in the ventral horn of WT mice by 3 mo of age. Strikingly, in two presymptomatic ALS mouse strains transgenic for mutant human Cu/Zn superoxide dismutase (SOD1), G85R SOD1YFP and G93A SOD1, little or no lipofuscin was detected in motor neuron cell bodies. Two markers of autophagy, sequestosome 1 (SQSTM1/p62) and microtubule-associated protein 1 light chain 3 (LC3), were examined in the motor neuron cell bodies of G85R SOD1YFP mice and found to be reduced relative to WT SOD1YFP transgenic mice. To elucidate whether the autophagy/lysosome pathway was either impaired or hyperactive in motor neurons, chloroquine was administered to 3-mo-old G85R SOD1YFP mice to block lysosomal hydrolysis. After 2 wk, lipofuscin was now observed in motor neurons, and SQSTM1 and LC3 levels approached those of WT SOD1YFP mice, suggesting that the autophagy/lysosome pathway is hyperactive in motor neurons of SOD1-linked ALS mice. This seems to be mediated at least in part through the mammalian target of rapamycin complex 1 (MTORC1) pathway, because levels of Ser757-phosphorylated Unc-51-like kinase 1 (ULK1), an MTORC1 target, were greatly reduced in the G85R SOD1YFP motor neurons, correspondent to an activated state of ULK1 that initiates autophagy.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Autofagia , Lipofuscina/metabolismo , Lisosomas/metabolismo , Neuronas Motoras/metabolismo , Superóxido Dismutasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Lipofuscina/genética , Lisosomas/genética , Lisosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas Motoras/patología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación Missense , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Sequestosoma-1 , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The early decades of Cell witnessed key discoveries that coalesced into the field of chaperones, protein folding, and protein quality control.
Asunto(s)
Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteínas/metabolismo , Animales , Escherichia coli/citología , Escherichia coli/metabolismo , Levaduras/citología , Levaduras/metabolismoRESUMEN
High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose-response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50 <10 µM). The screening results presented herein represent the first steps in a broader aim of developing molecular probes to study chaperonin biochemistry and physiology.
Asunto(s)
Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Modelos Biológicos , Pliegue de Proteína/efectos de los fármacosRESUMEN
Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Captura por Microdisección con Láser/métodos , Neuronas/citología , ARN/aislamiento & purificación , Médula Espinal/citología , Animales , Colorantes Azulados/química , Ratones , Neuronas/química , ARN/química , ARN/genética , Médula Espinal/químicaRESUMEN
We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.
Asunto(s)
Bioquímica/historia , Chaperoninas/metabolismo , Pliegue de Proteína , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Chaperoninas/química , Connecticut , Historia del Siglo XX , Historia del Siglo XXIRESUMEN
Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.
Asunto(s)
Transporte Axonal/fisiología , Proteínas del Choque Térmico HSP110/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Superóxido Dismutasa/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Decapodiformes , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Mutación Missense/genética , Fosforilación/efectos de los fármacos , Pliegue de Proteína , Proteómica , Médula Espinal/citología , Médula Espinal/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Vesículas Transportadoras/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1(GLU7ALA), compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1(GLU7ALA) relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.