RESUMEN
The heat shock protein 90 kDa (Hsp90) molecular chaperone machinery is responsible for the folding and activation of hundreds of important clients such as kinases, steroid hormone receptors, transcription factors, etc. This process is dynamically regulated in an ATP-dependent manner by Hsp90 co-chaperones including a group of tetratricopeptide (TPR) motif proteins that bind to the C-terminus of Hsp90. Among these TPR containing co-chaperones, FK506-binding protein 51 kDa (FKBP51) is reported to play an important role in stress-related pathologies, psychiatric disorders, Alzheimer's disease, and cancer, making FKBP51-Hsp90 interaction a potential therapeutic target. In this study, we report identification of potent and selective inhibitors of FKBP51-Hsp90 protein-protein interaction using a structure-based virtual screening approach. Upon in vitro evaluation, the identified hits show a considerable degree of selectivity towards FKBP51 over other TPR proteins, particularly for highly homologous FKBP52. Tyr355 of FKBP51 emerged as an important contributor to inhibitor's specificity. Additionally, we demonstrate the impact of these inhibitors on cellular energy metabolism, and neurite outgrowth, which are subjects of FKBP51 regulation. Overall, the results from this study highlight a novel pharmacological approach towards regulation of FKBP51 function and more generally, Hsp90 function via its interaction with TPR co-chaperones.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Proteínas de Unión a Tacrolimus , Humanos , Unión Proteica , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares , Factores de Transcripción/metabolismoRESUMEN
FK506-binding protein 51 kDa (FKBP51), encoded by Fkbp5 gene, gained considerable attention as an important regulator of several aspects of human biology including stress response, metabolic dysfunction, inflammation, and age-dependent neurodegeneration. Its catalytic peptidyl-prolyl isomerase (PPIase) activity is mediated by the N-terminal FK506-binding (FK1) domain, whereas the C-terminal tetratricopeptide motif (TPR) domain is responsible for FKBP51 interaction with molecular chaperone heat shock protein 90 (Hsp90). To understand FKBP51-related biology, several mouse models have been created. These include Fkbp5 complete and conditional knockouts, overexpression, and humanized models. To dissect the role of FKBP51-Hsp90 interaction in FKBP51 biology, we have created an interaction-deficient mouse (Fkbp5TPRmut) by introducing two-point mutations in the TPR domain of FKBP51. FKBP51-Hsp90 interaction-deficient mice are viable, fertile and show Mendelian inheritance. Intracellular association of FKBP51 with Hsp90 is significantly reduced in homozygous mutants compared to wild-type animals. No behavioral differences between genotypes were seen at 2 months of age, however, sex-dependent differences were detected in Y-maze and fear conditioning tests at the age of 12 months. Moreover, we have found a significant reduction in plasma levels of corticosterone and adrenocorticotropic hormone in Fkbp5TPRmut mice after acute stress. In contrast to Fkbp5 knockout mice, females of Fkbp5TPRmut showed increased body weight gain under high-fat diet treatment. Our data confirm the importance of FKBP51-Hsp90 interactions for stress-related endocrine signaling. Also, Fkbp5TPRmut mice can serve as a useful in vivo tool to discriminate between Hsp90-dependent and independent functions of FKBP51.
Asunto(s)
Dieta Alta en Grasa , Caracteres Sexuales , Animales , Femenino , Humanos , Lactante , Masculino , Ratones , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
The dysfunction of the proteostasis network is a molecular hallmark of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Molecular chaperones are a major component of the proteostasis network and maintain cellular homeostasis by folding client proteins, assisting with intracellular transport, and interfering with protein aggregation or degradation. Heat shock protein 70 kDa (Hsp70) and 90 kDa (Hsp90) are two of the most important chaperones whose functions are dependent on ATP hydrolysis and collaboration with their co-chaperones. Numerous studies implicate Hsp70, Hsp90, and their co-chaperones in neurodegenerative diseases. Targeting the specific protein-protein interactions between chaperones and their particular partner co-chaperones with small molecules provides an opportunity to specifically modulate Hsp70 or Hsp90 function for neurodegenerative diseases. Here, we review the roles of co-chaperones in Hsp70 or Hsp90 chaperone cycles, the impacts of co-chaperones in neurodegenerative diseases, and the development of small molecules modulating chaperone/co-chaperone interactions. We also provide a future perspective of drug development targeting chaperone/co-chaperone interactions for neurodegenerative diseases.
Asunto(s)
Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/genética , Humanos , Enfermedades Neurodegenerativas/patologíaRESUMEN
Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Unión ProteicaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia characterized by presence of extracellular amyloid plaques and intracellular neurofibrillary tangles composed of tau protein. Currently there are close to 50 million people living with dementia and this figure is expected to increase to 75 million by 2030 putting a huge burden on the economy due to the health care cost. Considering the effects on quality of life of patients and the increasing burden on the economy, there is an enormous need of new disease modifying therapies to tackle this disease. The current therapies are dominated by only symptomatic treatments including cholinesterase inhibitors and N-methyl-D-aspartate receptor blockers but no disease modifying treatments exist so far. After several failed attempts to develop drugs against amyloidopathy, tau targeting approaches have been in the main focus of drug development against AD. After an overview of the tauopathy in AD, this review summarizes recent findings on the development of small molecules as therapeutics targeting tau modification, aggregation, and degradation, and tau-oriented multi-target directed ligands. Overall, this work aims to provide a comprehensive and critical overview of small molecules which are being explored as a lead candidate for discovering drugs against tauopathy in AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Fármacos Neuroprotectores/química , Proteínas tau/metabolismo , Animales , Benzodioxoles/farmacología , Inhibidores de la Colinesterasa/farmacología , Colinesterasas/metabolismo , Curcumina/farmacología , Humanos , Terapia Molecular Dirigida , Ovillos Neurofibrilares/metabolismo , Fármacos Neuroprotectores/farmacología , Fosforilación , Placa Amiloide/metabolismo , Agregación Patológica de Proteínas/prevención & control , Procesamiento Proteico-Postraduccional , Quinazolinas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tiadiazoles/farmacologíaRESUMEN
Alzheimer's disease (AD) represents a major public health threat and, unfortunately, available therapeutics provide only temporary symptomatic relief. AD is a complex multifactorial disease and failure of single target therapeutics targeting amyloid-ß (Aß) in recent clinical trials suggests that future AD drug development should be focused on simultaneous targeting of several pathological hallmarks of the disease. Recently, we have shown that GMP-1, a 2-(methoxymethyl)pyrimido [1, 2-a] benzimidazol-4-ol, protects mitochondrial function in drosophila and mice models of AD, and improved memory and behavior indicating neuroprotective effect of GMP-1 treatment. Here, we have found that GMP-1 specifically binds to copper and zinc, metals that are dysregulated in AD brain. Addition of GMP-1 does not inhibit metal-dependent enzymatic reactions. Also, binding of Zn(II) and Cu(II) by GMP-1 is weaker than the 8-hydroxyquinoline scaffold compound clioquinol previously tested in AD clinical trials. However, GMP-1 affects Cu(II)-dependent Aß fibrillization as well as oxidative damage and viability of SH-SY5Y cells upon addition of Cu(II) and Aß. Our data provide new insight on GMP-1 as a Zn(II) and Cu(II) specific metal chelator of moderate affinity that can be responsible for some of its neuroprotective effects observed in AD animal models.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Bencimidazoles/metabolismo , Bencimidazoles/uso terapéutico , Quelantes/metabolismo , Quelantes/uso terapéutico , Metales/metabolismo , Enfermedades del Sistema Nervioso/inducido químicamente , Enfermedades del Sistema Nervioso/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/toxicidad , Pirimidinas/metabolismo , Pirimidinas/uso terapéutico , Bencimidazoles/farmacología , Muerte Celular , Línea Celular , Clioquinol/farmacología , Cobre/metabolismo , Humanos , Ligandos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Zinc/metabolismoRESUMEN
Dicarboxylate clamp tetratricopeptide repeat (dcTPR) motif-containing proteins are well-known partners of the heat shock protein (Hsp) 70 and Hsp90 molecular chaperones. Together, they facilitate a variety of intracellular processes, including protein folding and maturation, protein targeting, and protein degradation. An extreme C-terminal sequence, the EEVD motif, is identical in Hsp70 and Hsp90, and is indispensable for their interaction with dcTPR proteins. However, almost no information is available on the existence of other potential dcTPR-interacting proteins. We searched the human protein database for proteins with C-terminal sequences similar to that of Hsp70/Hsp90 to identify potential partners of dcTPR proteins. The search identified 112 proteins containing a Hsp70/Hsp90-like signature at their C termini. Gene Ontology enrichment analysis of identified proteins revealed enrichment of distinct protein classes, such as molecular chaperones and proteins of the ubiquitin-proteasome system, highlighting the possibility of functional specialization of proteins containing a Hsp70/Hsp90-like signature. We confirmed interactions of selected proteins containing Hsp70/Hsp90-like C termini with dcTPR proteins both in vitro and in situ. Analysis of interactions of 10-amino-acid peptides corresponding to the C termini of identified proteins with dcTPR proteins revealed significant differences in binding strength between various peptides. We propose a hierarchical mode of interaction within the dcTPR protein network. These findings describe a novel dcTPR protein interaction networks and provide a rationale for selective regulation of protein-protein interactions within this network.
RESUMEN
Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aß) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria-destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP-1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP-1 treatment of SH-SY5Y cells results in decrease in mitochondria-associated APP and protects SH-SY5Y cells from toxic effect of Aß1-42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP-1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Bencimidazoles/farmacología , Mitocondrias/efectos de los fármacos , Chaperonas Moleculares/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Animales Modificados Genéticamente , Conducta Animal/efectos de los fármacos , Bencimidazoles/uso terapéutico , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Simulación del Acoplamiento Molecular , Actividad Motora/efectos de los fármacos , Fragmentos de Péptidos/genética , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Cristalografía por Rayos X , Escherichia coli , Fluorometría , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Dominios Proteicos , TemperaturaRESUMEN
Accumulation of amyloid ß-peptide (Aß) in the brain is an important event in the pathogenesis of Alzheimer disease. We have used a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation to investigate whether Aß deposition is correlated with mitochondrial functions in these animals. We found evidence of mitochondrial dysfunction (i.e., decreased mitochondrial membrane potential, increased production of reactive oxygen species and oxidative DNA damage) at 6 months of age, when the mice showed very mild Aß deposition. More pronounced mitochondrial abnormalities were present in 24-month-old TgAPParc mice with more extensive Aß pathology. This study demonstrates for the first time mitochondrial dysfunction in transgenic mice with a mutation within the Aß peptide (the Arctic APP mutation), and confirms previous studies suggesting that mitochondrial dysfunction and oxidative stress is an early event in the pathogenesis of Alzheimer disease. This study demonstrates mitochondrial dysfunction in transgenic mice with a mutation within the amyloid beta (Aß) peptide (the Arctic amyloid precursor protein (APP) mutation). We found evidence of mitochondrial dysfunction (i.e. decreased mitochondrial membrane potential (MMP), increased production of reactive oxygen species (ROS) and oxidative DNA damage) at 6 months of age, when very mild Aß deposition is present in the mice. Also, the cytochrome c (COX) activity was significantly decreased in mitochondria from transgenic mice at 24 months of age.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Enfermedades Mitocondriales/genética , Mutación/genética , 8-Hidroxi-2'-Desoxicoguanosina , Factores de Edad , Péptidos beta-Amiloides/metabolismo , Análisis de Varianza , Animales , Encéfalo/metabolismo , Encéfalo/patología , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/fisiopatología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Previous findings demonstrated an altered pattern of amyloid-ß protein precursor (AßPP) expression in platelets of Alzheimer's disease (AD) patients compared with either healthy control subjects or patients with non-Alzheimer-type dementia. In an attempt to explore the diagnostic potential of platelet AßPP metabolism, we have generated monoclonal antibodies directed to the N-terminal part of AßPP. We have observed two different antibody recognition patterns of AßPP: one resembling previously described 130 kDa and 105 kDa species and a novel AßPP 115 kDa form. This form was significantly increased in platelets of the mild cognitive impairment and AD group as compared to control subjects. The abundance of AßPP 115 kDa species correlated with the previously described AßPP 130/105 kDa ratio as well as with Mini-Mental State Examination score. Despite the inability of these particular monoclonal antibodies to recognize native forms of AßPP, identification of a new AßPP isoform in platelets as a potential AD biomarker can provide an additional tool for the development of a reliable diagnostic test to detect preclinical stages of AD.
Asunto(s)
Enfermedad de Alzheimer/sangre , Precursor de Proteína beta-Amiloide/sangre , Biomarcadores/sangre , Plaquetas/metabolismo , Anciano , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Anticuerpos Monoclonales/química , Apolipoproteína E4/genética , Plaquetas/química , Western Blotting , Línea Celular , Disfunción Cognitiva/genética , Disfunción Cognitiva/psicología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Glicosilación , Humanos , Inmunoprecipitación , Isomerismo , Masculino , Ratones , Pruebas Neuropsicológicas , Activación Plaquetaria , Estándares de ReferenciaRESUMEN
Intracellular amyloid-ß peptide (Aß) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aß that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aß are produced locally and may contribute to mitochondrial dysfunction in AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Western Blotting , Carbamatos/farmacología , Línea Celular Tumoral , Células Cultivadas , Dipéptidos/farmacología , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Especificidad por SustratoRESUMEN
Omi/HtrA2, a mitochondrial serine protease with chaperone activity, is involved in varied intracellular processes. Dysfunctional Omi/HtrA2 has thus been implicated in various neurodegenerative disorders. Previously, we have shown that γ-secretase complexes are present and active in mitochondria. Here, we demonstrate that peptide corresponding to C-terminus of presenilin-1, as previously reported to activate Omi/HtrA2, interacts with Omi/HtrA2 in isolated mitochondria. Moreover, we show that Omi/HtrA2 interacts with presenilin in active γ-secretase complexes located to mitochondria. Using a biotinylated γ-secretase inhibitor and confocal microscopy, we could further confirm the association of γ-secretase complexes with mitochondrial Omi/HtrA2. Furthermore, determination of γ-secretase complex topology in isolated mitochondria revealed an association of γ-secretase complexes with the outer membrane of mitochondria with the extreme PS1 C-terminus facing the inter-membrane space. We have also studied the impact of Omi/HtrA2 on γ-secretase activity, measuring APP intracellular domain (AICD) production. We found reduced AICD production in mitochondria isolated from Omi/HtrA2 knockout mouse embryonic fibroblasts, indicating a significant role of Omi/HtrA2 on γ-secretase activity. Thus, our results provide information for understanding the interplay between mitochondrial Omi/HtrA2 and γ-secretase complexes in AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Serina Peptidasa A2 que Requiere Temperaturas Altas , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitochondrial function of neurons within specific brain regions. This is accompanied by an elevated production of reactive oxygen species contributing to increased rates of neuronal loss in the AD-affected brain regions. In this review, we will discuss the role of mitochondrial function and dysfunction in AD. We will focus on the consequences of amyloid precursor protein and amyloid-beta peptide accumulation in mitochondria and their involvement in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Mitocondrias/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/toxicidad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Especificidad de ÓrganosRESUMEN
The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Péptidos/metabolismo , Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/ultraestructura , Animales , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Endopeptidasa K/farmacología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Microscopía Inmunoelectrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Neuroblastoma/metabolismo , Péptidos/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
Asunto(s)
Mitocondrias/metabolismo , Biología Molecular/métodos , Plantas/metabolismo , Proteínas/metabolismo , Cloroplastos/metabolismo , Electroporación , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Modelos Biológicos , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/metabolismo , Spinacia oleracea/metabolismoRESUMEN
In plants, the majority of mitochondrial and chloroplast proteins are nuclear encoded, synthesized on cytosolic polyribosomes, and then imported into the organelle. Most of the nuclear encoded precursor proteins contain an N-terminal extension called signal or targeting peptide that directs the protein to the correct organelle. Here, we describe in vitro and in vivo methods to study mitochondrial protein import. In a common single-organelle in vitro import procedure, transcribed/translated precursor proteins are imported into isolated mitochondria. A novel semi-in vivo system for simultaneous import of precursor proteins into isolated mitochondria and chloroplasts, called a dual-import system, is superior to the single-import system as it abolishes mistargeting of chloroplast precursors into mitochondria as observed in a single-organelle import system. Precursor proteins can also be imported into the organelles in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a targeting peptide or a precursor protein fused to a reporter gene, most commonly the green fluorescence protein in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
Asunto(s)
Mitocondrias/metabolismo , Biología Molecular/métodos , Plantas/metabolismo , Cloroplastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/metabolismo , Spinacia oleracea/metabolismoRESUMEN
Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.
Asunto(s)
Péptidos beta-Amiloides/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/fisiología , Péptidos/química , Serina Endopeptidasas/fisiología , Partículas Submitocóndricas/química , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Animales , Corteza Cerebral/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/química , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/químicaRESUMEN
Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the F(A)d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F(A)d protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the F(A)d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F(A)d (pAOX-mF(A)d) could be imported. Import competence of F(A)d and pAOX-mF(A)d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F(A)d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.
Asunto(s)
Proteínas Mitocondriales/genética , Biosíntesis de Proteínas/genética , Transcripción Genética/genética , Proteínas 14-3-3 , Animales , Sistema Libre de Células/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Extractos Vegetales/genética , Extractos Vegetales/farmacología , Pruebas de Precipitina , Unión Proteica , Pliegue de Proteína , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Conejos , Reticulocitos/metabolismo , Solubilidad , Triticum/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Tim44 is an essential component of the translocase of the inner mitochondrial membrane (TIM) complex that mediates transport of nuclear encoded mitochondrial precursors across the inner membrane. Here, we have investigated the topology of Tim44 by probing mitochondria with membrane impermeable 3-(N-maleimidopropionyl)biocytin (MPB) followed by the specific immunoprecipitation of modified proteins. Our data indicate that a single cysteine residue, Cys-369, located in the C-terminal domain of the yeast Tim44 is exposed to the mitochondrial intermembrane space.