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1.
Am J Hum Genet ; 108(10): 1964-1980, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34547244

RESUMO

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.


Assuntos
Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/fisiologia , Anormalidades Craniofaciais/genética , Variações do Número de Cópias de DNA , Anormalidades do Olho/genética , Transtornos do Crescimento/genética , Hérnias Diafragmáticas Congênitas/genética , Luxação Congênita de Quadril/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Mutação de Sentido Incorreto , Osteocondrodisplasias/genética , Anormalidades Dentárias/genética , Animais , Estudos de Casos e Controles , Estudos de Coortes , Anormalidades Craniofaciais/patologia , Anormalidades do Olho/patologia , Feminino , Transtornos do Crescimento/patologia , Hérnias Diafragmáticas Congênitas/patologia , Luxação Congênita de Quadril/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteocondrodisplasias/patologia , Linhagem , Anormalidades Dentárias/patologia
2.
J Biol Chem ; 297(4): 101134, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461102

RESUMO

The mitochondrial matrix protease LONP1 is an essential part of the organellar protein quality control system. LONP1 has been shown to be involved in respiration control and apoptosis. Furthermore, a reduction in LONP1 level correlates with aging. Up to now, the effects of a LONP1 defect were mostly studied by utilizing transient, siRNA-mediated knockdown approaches. We generated a new cellular model system for studying the impact of LONP1 on mitochondrial protein homeostasis by a CRISPR/Cas-mediated genetic knockdown (gKD). These cells showed a stable reduction of LONP1 along with a mild phenotype characterized by absent morphological differences and only small negative effects on mitochondrial functions under normal culture conditions. To assess the consequences of a permanent LONP1 depletion on the mitochondrial proteome, we analyzed the alterations of protein levels by quantitative mass spectrometry, demonstrating small adaptive changes, in particular with respect to mitochondrial protein biogenesis. In an additional proteomic analysis, we determined the temperature-dependent aggregation behavior of mitochondrial proteins and its dependence on a reduction of LONP1 activity, demonstrating the important role of the protease for mitochondrial protein homeostasis in mammalian cells. We identified a significant number of mitochondrial proteins that are affected by a reduced LONP1 activity especially with respect to their stress-induced solubility. Taken together, our results suggest a very good applicability of the LONP1 gKD cell line as a model system for human aging processes.


Assuntos
Proteases Dependentes de ATP/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Agregados Proteicos , Proteoma/metabolismo , Proteômica , Proteases Dependentes de ATP/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteoma/genética
3.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445316

RESUMO

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.


Assuntos
Ácido Dicloroacético/farmacologia , Inibidores Enzimáticos/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Linhagem Celular Tumoral , Endopeptidase Clp/antagonistas & inibidores , Endopeptidase Clp/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Células PC-3 , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Ratos
4.
J Neurol Sci ; 428: 117600, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34333379

RESUMO

The AFG3L2 gene encodes AFG3-like protein 2, which is a subunit of human mitochondrial ATPases associated with various cellular protease activities (m-AAA). The clinical spectrum of AFG3L2 mutations is broad. Dominant AFG3L2 mutations can cause autosomal dominant spinocerebellar ataxia type 28 (SCA28), whereas biallelic AFG3L2 mutations may lead to spastic ataxia 5 (SPAX5). However, the role of AFG3L2 mutations in autosomal recessive spinocerebellar ataxia (SCAR) remains elusive. The aim of this study is to delineate the clinical features and spectrum of AFG3L2 mutations in a Taiwanese cohort with cerebellar ataxia. Mutational analyses of AFG3L2 were carried out by targeted resequencing in a cohort of 133 unrelated patients with molecularly undetermined cerebellar ataxia. We identified one single patient carrying compound heterozygous mutations in AFG3L2, p.[R632*];[V723M] (c.[1894C > T];[2167G > A]). The patient has suffered from apparently sporadic and slowly progressive cerebellar ataxia, ptosis, and ophthalmoparesis since age 55 years. These findings expand the clinical spectrum of AFG3L2 mutations and suggest a new subtype of late-onset SCAR caused by biallelic AFG3L2 mutations.


Assuntos
Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Mutação de Sentido Incorreto , Ataxias Espinocerebelares , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Humanos , Pessoa de Meia-Idade , Fenótipo , Ataxias Espinocerebelares/genética
6.
Nat Commun ; 12(1): 3239, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050165

RESUMO

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.


Assuntos
Proteases Dependentes de ATP/ultraestrutura , Proteínas Mitocondriais/ultraestrutura , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Bortezomib/farmacologia , Domínio Catalítico/efeitos dos fármacos , Microscopia Crioeletrônica , Ensaios Enzimáticos , Humanos , Hidrólise , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Proteólise , Proteostase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
7.
Nucleic Acids Res ; 49(9): 5230-5248, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33956154

RESUMO

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.


Assuntos
DNA Polimerase gama/genética , Proteases Dependentes de ATP/metabolismo , Animais , Células Cultivadas , DNA Polimerase gama/metabolismo , Replicação do DNA , DNA Mitocondrial/análise , Estabilidade Enzimática/genética , Células HeLa , Holoenzimas/metabolismo , Humanos , Camundongos , Proteínas Mitocondriais/metabolismo , Mutação
8.
Sci Rep ; 11(1): 7320, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795807

RESUMO

DJ-1/PARK7 mutations are linked with familial forms of early-onset Parkinson's disease (PD). We have studied the degradation of untagged DJ-1 wild type (WT) and missense mutants in mouse embryonic fibroblasts obtained from DJ-1-null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants L10P, M26I, A107P, P158Δ, L166P, E163K, and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E, and A179T are as stable as DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158Δ, L166P, E163K, and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of the mitochondrial-associated DJ-1 mutants A107P, P158Δ, L166P, E163K, and L172Q but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis.


Assuntos
Proteases Dependentes de ATP/biossíntese , Mitocôndrias/enzimologia , Proteínas Mitocondriais/biossíntese , Mutação de Sentido Incorreto , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteína Desglicase DJ-1/genética , Animais , Fibroblastos/metabolismo , Inativação Gênica , Homozigoto , Humanos , Camundongos , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma , Frações Subcelulares
9.
J Med Chem ; 64(8): 4857-4869, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33821636

RESUMO

LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.


Assuntos
Proteases Dependentes de ATP/antagonistas & inibidores , Desenho de Fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Inibidores de Proteases/química , Proteases Dependentes de ATP/metabolismo , Sítios de Ligação , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib/química , Bortezomib/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
10.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668863

RESUMO

Lon protease 1 (LONP1) is a highly conserved serine peptidase that plays an important role in the protein quality control system in mammalian mitochondria. LONP1 catalyzes the degradation of oxidized, dysfunctional, and misfolded matrix proteins inside mitochondria and regulates mitochondrial gene expression and genome integrity. Therefore, LONP1 is up-regulated and suppresses cell death in response to oxidative stress, heat shock, and nutrient starvation. On the other hand, translocation of high mobility group box 1 (HMGB1) and active caspase-3 into mitochondria is involved in apoptosis of parvalbumin (PV) cells (one of the GABAergic interneurons) and necrosis of CA1 neurons in the rat hippocampus, respectively, following status epilepticus (SE). In the present study, we investigated whether LONP1 may improve neuronal viability to prevent or ameliorate translocation of active caspase-3 and HMGB1 in mitochondria within PV and CA1 neurons. Following SE, LONP1 expression was up-regulated in mitochondria of PV and CA1 neurons. LONP1 knockdown deteriorated SE-induced neuronal death with mitochondrial accumulation of active caspase-3 and HMGB1 in PV cells and CA1 neurons, respectively. LONP1 knockdown did not affect the aberrant mitochondrial machinery induced by SE. Therefore, our findings suggest, for the first time, that LONP1 may contribute to the alleviation of mitochondrial overloads of active caspase-3 and HMGB1, and the maintenance of neuronal viability against SE.


Assuntos
Proteases Dependentes de ATP/metabolismo , Região CA1 Hipocampal/patologia , Caspase 3/metabolismo , Proteína HMGB1/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Parvalbuminas/metabolismo , Animais , Morte Celular , Técnicas de Silenciamento de Genes , Masculino , Dinâmica Mitocondrial , Pilocarpina , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Estado Epiléptico
11.
Mov Disord ; 36(7): 1664-1675, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33598982

RESUMO

BACKGROUND: Although the typical inheritance of spastic paraplegia 7 is recessive, several reports have suggested that SPG7 variants may also cause autosomal dominant hereditary spastic paraplegia (HSP). OBJECTIVES: We aimed to conduct an exome-wide genetic analysis on a large Canadian cohort of HSP patients and controls to examine the association of SPG7 and HSP. METHODS: We analyzed 585 HSP patients from 372 families and 1175 controls, including 580 unrelated individuals. Whole-exome sequencing was performed on 400 HSP patients (291 index cases) and all 1175 controls. RESULTS: The frequency of heterozygous pathogenic/likely pathogenic SPG7 variants (4.8%) among unrelated HSP patients was higher than among unrelated controls (1.7%; OR 2.88, 95% CI 1.24-6.66, P = 0.009). The heterozygous SPG7 p.(Ala510Val) variant was found in 3.7% of index patients versus 0.85% in unrelated controls (OR 4.42, 95% CI 1.49-13.07, P = 0.005). Similar results were obtained after including only genetically-undiagnosed patients. We identified four heterozygous SPG7 variant carriers with an additional pathogenic variant in known HSP genes, compared to zero in controls (OR 19.58, 95% CI 1.05-365.13, P = 0.0031), indicating potential digenic inheritance. We further identified four families with heterozygous variants in SPG7 and SPG7-interacting genes (CACNA1A, AFG3L2, and MORC2). Of these, there is especially compelling evidence for epistasis between SPG7 and AFG3L2. The p.(Ile705Thr) variant in AFG3L2 is located at the interface between hexamer subunits, in a hotspot of mutations associated with spinocerebellar ataxia type 28 that affect its proteolytic function. CONCLUSIONS: Our results provide evidence for complex inheritance in SPG7-associated HSP, which may include recessive and possibly dominant and digenic/epistasis forms of inheritance. © 2021 International Parkinson and Movement Disorder Society.


Assuntos
Paraplegia Espástica Hereditária , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares/genética , Canadá , Humanos , Metaloendopeptidases/genética , Mutação/genética , Paraplegia , Paraplegia Espástica Hereditária/genética , Fatores de Transcrição
12.
Nat Commun ; 12(1): 265, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431889

RESUMO

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding. We show that LONP1, an AAA+ protease of the mitochondrial matrix, works with the mtHSP70 chaperone system to promote mitochondrial protein folding. Inhibition of LONP1 results in aggregation of a protein subset similar to that caused by knockdown of DNAJA3, a co-chaperone of mtHSP70. LONP1 is required for DNAJA3 and mtHSP70 solubility, and its ATPase, but not its protease activity, is required for this function. In vitro, LONP1 shows an intrinsic chaperone-like activity and collaborates with mtHSP70 to stabilize a folding intermediate of OXA1L. Our results identify LONP1 as a critical factor in the mtHSP70 folding pathway and demonstrate its proposed chaperone activity.


Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dobramento de Proteína , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons , Proteínas de Choque Térmico HSP40 , Humanos , NADH Desidrogenase , Proteínas Nucleares , Agregados Proteicos , Ligação Proteica , Transdução de Sinais , Solubilidade
13.
J Exp Bot ; 72(9): 3455-3473, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33216923

RESUMO

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.


Assuntos
Proteases Dependentes de ATP , Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Proteínas de Membrana , Proteases Dependentes de ATP/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Proteínas de Membrana/genética , Metaloproteases/genética , Mutação
14.
Biochim Biophys Acta Biomembr ; 1863(2): 183526, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33278347

RESUMO

FtsH is a membrane-bound protease that plays a crucial role in proteolytic regulation of many cellular functions. It is universally conserved in bacteria and responsible for the degradation of misfolded or misassembled proteins. A recent study has determined the structure of bacterial FtsH in detergent micelles. To properly study the function of FtsH in a native-like environment, we reconstituted the FtsH complex into lipid nanodiscs. We found that FtsH in membrane scaffold protein (MSP) nanodiscs maintains its native hexameric conformation and is functionally active. We further investigated the effect of the lipid bilayer composition (acyl chain length, saturation, head group charge and size) on FtsH proteolytic activity. We found that the lipid acyl chain length influences AaFtsH activity in nanodiscs, with the greatest activity in a bilayer of di-C18:1 PC. We conclude that MSP nanodiscs are suitable model membranes for further in vitro studies of the FtsH protease complex.


Assuntos
Proteases Dependentes de ATP/química , Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Nanoestruturas/química , Dobramento de Proteína , Aquifex/enzimologia , Aquifex/genética , Proteínas de Bactérias/genética
15.
Methods Mol Biol ; 2192: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230781

RESUMO

Mitochondrial proteases constitute a fundamental part of the organellar protein quality control system to ensure the timely removal of damaged or obsolete proteins. The analysis of proteases is often limited to the identification of bona fide substrates that are degraded in the presence and become more abundant in the absence of the respective protease. However, proteases in numerous organisms from bacteria to humans can process specific substrates to release shortened proteins with potentially altered activities. Here, we describe an adaptation of the substrate-trapping approach, as well as the N-terminal profiling protocol Terminal Amine Isotope Labeling of Substrates (TAILS) for the identification of bona fide substrates and mitochondrial proteins that undergo complete or partial proteolysis.


Assuntos
Proteases Dependentes de ATP/química , Proteases Dependentes de ATP/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/genética , Animais , Células Cultivadas , Cromatografia Líquida/métodos , Fibroblastos/citologia , Imunoprecipitação/métodos , Marcação por Isótopo/métodos , Camundongos , Camundongos Knockout , Miocárdio/citologia , Processamento de Proteína Pós-Traducional , Proteólise , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos , Transfecção/métodos
16.
Int J Mol Sci ; 21(23)2020 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-33260377

RESUMO

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.


Assuntos
Amidoidrolases/metabolismo , Biocatálise , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/biossíntese , Proteases Dependentes de ATP/química , Proteases Dependentes de ATP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise/efeitos dos fármacos , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lipopolissacarídeos/química , Mutação/genética , Óperon/genética , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Fosfolipídeos/biossíntese , Fosfolipídeos/química , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteólise/efeitos dos fármacos , Supressão Genética , Temperatura , Treonina/análogos & derivados , Treonina/farmacologia , Transcrição Genética/efeitos dos fármacos
17.
Nucleic Acids Res ; 48(21): 12030-12041, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33211866

RESUMO

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.


Assuntos
Proteases Dependentes de ATP/genética , Colífagos/genética , Endopeptidases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lisogenia , Proteínas de Membrana/genética , Prófagos/genética , Proteínas Virais/genética , Proteases Dependentes de ATP/metabolismo , Colífagos/crescimento & desenvolvimento , Colífagos/metabolismo , Colífagos/efeitos da radiação , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Modelos Estatísticos , Prófagos/crescimento & desenvolvimento , Prófagos/metabolismo , Prófagos/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Processos Estocásticos , Ativação Transcricional , Raios Ultravioleta , Proteínas Virais/metabolismo
18.
PLoS Genet ; 16(10): e1009118, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33075064

RESUMO

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2.


Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Transporte de Elétrons/genética , Mitocôndrias/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Peptídeo Hidrolases , Pupa/genética , Pupa/crescimento & desenvolvimento , Interferência de RNA , Ribossomos/genética
19.
Am J Physiol Cell Physiol ; 319(6): C1020-C1028, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32936696

RESUMO

Myoblast differentiation is a crucial process for myogenesis. Mitochondria function as an energy-providing machine that is critical to this process, and mitochondrial dysfunction can prevent myoblasts from fusing into myotubes. However, the molecular mechanisms underlying the dynamic regulation of mitochondrial networks remain poorly understood. In the present study, we found that the PTEN induced kinase 1 (PINK1)/Parkin (an E3 ubiquitin-protein ligase) pathway is activated at the early stage of myoblast differentiation. Moreover, downregulation of mitofusin 2 (Mfn2) and increased dynamin-related protein 1 (Drp1) resulted in loosely formed mitochondria during this period. Furthermore, selective knockdown of the mitochondrial matrix protein Lon peptidase-1 (LonP1) at the early stage of myoblast differentiation induced mitochondrial depolarization and suppressed the PINK1/Parkin pathway and reduced Mfn2 and Drp1 levels, which blocked mitochondrial remodeling and myoblast differentiation. Overall, these data demonstrate that LonP1 promotes myoblast differentiation by regulating PINK1/Parkin-mediated mitochondrial remodeling.


Assuntos
Proteases Dependentes de ATP/metabolismo , Diferenciação Celular/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mioblastos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Desenvolvimento Muscular/fisiologia
20.
Acta Neuropathol Commun ; 8(1): 93, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600459

RESUMO

Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.


Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Domínio AAA/genética , Adolescente , Criança , Pré-Escolar , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Metaloendopeptidases/metabolismo , Mutação de Sentido Incorreto , Linhagem
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