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1.
Elife ; 122024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38900507

RESUMO

Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the 'mitochondria as guardian in cytosol' (MAGIC) whereby cytosolic misfolded proteins (MPs) are imported into and degraded inside mitochondria. In this study, a genome-wide screen in Saccharomyces cerevisiae uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of MPs into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by MPs such as those associated with neurodegenerative diseases.


Assuntos
Mitocôndrias , Dobramento de Proteína , Transporte Proteico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Glucose/metabolismo
2.
NPJ Parkinsons Dis ; 10(1): 120, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38906862

RESUMO

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by mitochondrial dysfunction and accumulation of alpha-synuclein (α-Syn)-containing protein aggregates known as Lewy bodies (LB). Here, we investigated the entry of α-Syn into mitochondria to cause mitochondrial dysfunction and loss of cellular fitness in vivo. We show that α-Syn expressed in yeast and human cells is constitutively imported into mitochondria. In a transgenic mouse model, the level of endogenous α-Syn accumulation in mitochondria of dopaminergic neurons and microglia increases with age. The imported α-Syn is degraded by conserved mitochondrial proteases, most notably NLN and PITRM1 (Prd1 and Cym1 in yeast, respectively). α-Syn in the mitochondrial matrix that is not degraded interacts with respiratory chain complexes, leading to loss of mitochondrial DNA (mtDNA), mitochondrial membrane potential and cellular fitness decline. Importantly, enhancing mitochondrial proteolysis by increasing levels of specific proteases alleviated these defects in yeast, human cells, and a PD model of mouse primary neurons. Together, our results provide a direct link between α-synuclein-mediated cellular toxicity and its import into mitochondria and reveal potential therapeutic targets for the treatment of α-synucleinopathies.

3.
Sci Adv ; 10(14): eadj7666, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38569041

RESUMO

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.


Assuntos
Doenças Inflamatórias Intestinais , Prostaglandinas , Humanos , Epitélio/metabolismo , Inflamação , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Fibroblastos/metabolismo
4.
G3 (Bethesda) ; 14(3)2024 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-38289859

RESUMO

The decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation through the "mitochondrial as guardian in cytosol" (MAGIC) pathway. Here, we report an unexpected role of Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored ß-1,3-glucanosyltransferase in the budding yeast, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of GAS1 inhibits MAGIC but elevates protein ubiquitination and UPS-mediated protein degradation. Interestingly, we found that the Gas1 protein exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins through MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1-E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.


Assuntos
Proteostase , Saccharomycetales , Glicosilfosfatidilinositóis/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Saccharomycetales/metabolismo
5.
bioRxiv ; 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37808771

RESUMO

Inflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed an in vitro model whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFs in vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury.

6.
bioRxiv ; 2023 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-37292646

RESUMO

Decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation via 'mitochondrial as guardian in cytosol' (MAGIC) pathway. Here we report an unexpected role of yeast Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored ß-1,3-glucanosyltransferase, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of Gas1 inhibits MAGIC but elevates polyubiquitination and UPS-mediated protein degradation. Interestingly, we found that Gas1 exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins via MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

7.
bioRxiv ; 2023 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-37034811

RESUMO

Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the 'mitochondria as guardian in cytosol' (MAGIC) whereby cytosolic misfolded proteins (MPs) are imported into and degraded inside mitochondria. In this study, a genome-wide screen in yeast uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of MPs into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by MPs such as those associated with neurodegenerative diseases.

8.
PLoS Biol ; 20(10): e3001839, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36269765

RESUMO

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.


Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Saccharomyces cerevisiae , Humanos , Ligação Proteica , Proteínas de Choque Térmico HSP70/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Processamento de Proteína Pós-Traducional , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo
9.
Sci Adv ; 6(32): eabc7288, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32821848

RESUMO

Proteostasis declines with age, characterized by the accumulation of unfolded or damaged proteins. Recent studies suggest that proteins constituting pathological inclusions in neurodegenerative diseases also enter and accumulate in mitochondria. How unfolded proteins are managed within mitochondria remains unclear. Here, we found that excessive unfolded proteins in the mitochondrial matrix of yeast cells are consolidated into solid-phase inclusions, which we term deposits of unfolded mitochondrial proteins (DUMP). Formation of DUMP occurs in mitochondria near endoplasmic reticulum-mitochondria contact sites and is regulated by mitochondrial proteins controlling the production of cytidine 5'-diphosphate-diacylglycerol. DUMP formation is age dependent but accelerated by exogenous unfolded proteins. Many enzymes of the tricarboxylic acid cycle were enriched in DUMP. During yeast cell division, DUMP formation is necessary for asymmetric inheritance of damaged mitochondrial proteins between mother and daughter cells. We provide evidence that DUMP-like structures may be induced by excessive unfolded proteins in human cells.

10.
Nat Cell Biol ; 22(6): 689-700, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32313104

RESUMO

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.


Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Annu Rev Biophys ; 49: 41-67, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31928428

RESUMO

Mitochondria are essential organelles in eukaryotes. Most mitochondrial proteins are encoded by the nuclear genome and translated in the cytosol. Nuclear-encoded mitochondrial proteins need to be imported, processed, folded, and assembled into their functional states. To maintain protein homeostasis (proteostasis), mitochondria are equipped with a distinct set of quality control machineries. Deficiencies in such systems lead to mitochondrial dysfunction, which is a hallmark of aging and many human diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer. In this review, we discuss the unique challenges and solutions of proteostasis in mitochondria. The import machinery coordinates with mitochondrial proteases and chaperones to maintain the mitochondrial proteome. Moreover, mitochondrial proteostasis depends on cytosolic protein quality control mechanisms during crises. In turn, mitochondria facilitate cytosolic proteostasis. Increasing evidence suggests that enhancing mitochondrial proteostasis may hold therapeutic potential to protect against protein aggregation-associated cellular defects.


Assuntos
Mitocôndrias/metabolismo , Proteostase , Núcleo Celular/metabolismo , Citosol/metabolismo , Humanos , Proteínas Mitocondriais/metabolismo
12.
J Cell Sci ; 131(3)2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29420249

RESUMO

Aging is the gradual decline of physiological functions and organismal fitness, which leads to age-dependent fitness loss, diseases and eventually mortality. Understanding the cause of aging constitutes one of most intriguing areas of research in biology. On both the cellular and molecular levels, it has been hypothesized that there are aging determinants to control the onset and progression of aging, including the loss of beneficial components and accumulation of detrimental factors. This Review highlights the recent advance in identifying various factors that affect the aging process, focusing on how these determinants affect the lifespan and fitness of a cell or organism. With more and more aging determinants revealed, further understanding about their functions and interconnections could enable the development of specific intervention to extend healthy lifespan and reduce the risk of age-related diseases.


Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Senescência Celular , Humanos , Modelos Biológicos , Organelas/metabolismo
13.
Nature ; 543(7645): 443-446, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28241148

RESUMO

Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.


Assuntos
Citosol/metabolismo , Homeostase , Mitocôndrias/metabolismo , Agregados Proteicos/fisiologia , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Saccharomyces cerevisiae , Linhagem Celular , Citosol/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Redobramento de Proteína , Estabilidade Proteica , Transporte Proteico/efeitos dos fármacos , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
14.
Hum Mol Genet ; 23(25): 6863-77, 2014 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-25113748

RESUMO

TDP-43 proteinopathies are clinically and genetically heterogeneous diseases that had been considered distinct from classical amyloid diseases. Here, we provide evidence for the structural similarity between TDP-43 peptides and other amyloid proteins. Atomic force microscopy and electron microscopy examination of peptides spanning a previously defined amyloidogenic fragment revealed a minimal core region that forms amyloid fibrils similar to the TDP-43 fibrils detected in FTLD-TDP brain tissues. An ALS-mutant A315E amyloidogenic TDP-43 peptide is capable of cross-seeding other TDP-43 peptides and an amyloid-ß peptide. Sequential Nuclear Overhauser Effects and double-quantum-filtered correlation spectroscopy in nuclear magnetic resonance (NMR) analyses of the A315E-mutant TDP-43 peptide indicate that it adopts an anti-parallel ß conformation. When added to cell cultures, the amyloidogenic TDP-43 peptides induce TDP-43 redistribution from the nucleus to the cytoplasm. Neuronal cultures in compartmentalized microfluidic-chambers demonstrate that the TDP-43 peptides can be taken up by axons and induce axonotoxicity and neuronal death, thus recapitulating key neuropathological features of TDP-43 proteinopathies. Importantly, a single amino acid change in the amyloidogenic TDP-43 peptide that disrupts fibril formation also eliminates neurotoxicity, supporting that amyloidogenesis is critical for TDP-43 neurotoxicity.


Assuntos
Peptídeos beta-Amiloides/química , Córtex Cerebral/efeitos dos fármacos , Proteínas de Ligação a DNA/toxicidade , Neurônios/efeitos dos fármacos , Proteinopatias TDP-43/metabolismo , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/síntese química , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Técnicas Analíticas Microfluídicas , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Ratos , Proteinopatias TDP-43/patologia
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