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1.
Anticancer Res ; 42(1): 471-482, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34969757

RESUMO

BACKGROUND/AIM: HDAC6, a cytoplasmic localized deacetylase, is a positive regulator of cancer progression via modification of various substrates. We evaluated how the interaction between HDAC6 and glucose regulatory protein 78 (GRP78) affects the growth of cholangiocarcinoma (CCA). MATERIALS AND METHODS: The anti-tumor effects of ACY-1215, an HDAC6 specific inhibitor, in CCA cell lines were analyzed by cell viability assay, western blotting, flow cytometry, co-immunoprecipitation, and biotinylation assays. In vivo effects of ACY-1215 were evaluated in a xenograft model using CCA cell line TFK-1. RESULTS: ACY-1215 increased the acetyl-form of GRP78 by approximately 50% compared to control, which impaired the translocation of GRP78 to the plasma membrane by 50% through alteration of cellular proliferative signaling via PI3K/AKT. Furthermore, ACY-1215 suppressed tumor growth by 50% compared to vehicle control in a CCA xenograft model. CONCLUSION: Increase in GRP78 acetylation by HDAC6 inhibition suppressed GRP78 translocation to the cell surface, which inhibited proliferation and promoted apoptosis in CCA.


Assuntos
Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Desacetilase 6 de Histona/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Citometria de Fluxo , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/genética , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34747980

RESUMO

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.


Assuntos
Macroautofagia , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Sanguíneas/farmacologia , Células HeLa , Humanos , Células Jurkat , Lipoproteínas/metabolismo , Macroautofagia/efeitos dos fármacos , Peroxinas/metabolismo , Peroxissomos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ubiquitinação
3.
Methods Mol Biol ; 2303: 25-36, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626367

RESUMO

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.


Assuntos
Células Endoteliais , Linhagem Celular , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Transporte Proteico , Proteoglicanas/metabolismo
4.
Vet Microbiol ; 264: 109304, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34922148

RESUMO

DEAD (Asp-Glu-Ala-Asp)-box RNA helicases (DDX) play important roles in viral infection, either as cytosolic viral nucleic acids sensors or as essential host factors for viral replication. In this study, we identified DDX56 as a positive regulator for encephalomyocarditis virus (EMCV) replication. EMCV infection promotes DDX56 expression via its viral proteins, VP3 and 3C. We showed that DDX56 overexpression promotes EMCV replication whereas its loss dampened EMCV replication. Consequently, knockdown of DDX56 increases type I interferon (IFN) expression during EMCV infection. We also showed that DDX56 interrupts IFN regulatory factor 3 (IRF3) phosphorylation and its nucleus translocation by directly targeting KPNA3 and KPNA4 in an EMCV-triggered MDA5 signaling activation cascade leading to the blockade of IFN-ß production. Overall, we showed that DDX56 is a novel negative regulator of EMCV-mediated IFN-ß responses and that DDX56 plays a critical role in EMCV replication. These findings reveal a novel strategy for EMCV to utilize a host factor to evade the host innate immune response and provide us new insight into the function of DDX56.


Assuntos
RNA Helicases DEAD-box , Vírus da Encefalomiocardite , Interações Hospedeiro-Patógeno , Fator Regulador 3 de Interferon , Interferon beta , Transporte Proteico , Replicação Viral , Infecções por Cardiovirus/fisiopatologia , Infecções por Cardiovirus/virologia , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Vírus da Encefalomiocardite/fisiologia , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Replicação Viral/genética
5.
Biomolecules ; 11(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34944441

RESUMO

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.


Assuntos
Aldeído-Desidrogenase Mitocondrial/genética , Aldeídos/efeitos adversos , Dor/metabolismo , Proteína Quinase C-épsilon/metabolismo , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Carragenina/efeitos adversos , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Masculino , Camundongos , Mitocôndrias/metabolismo , Dor/induzido quimicamente , Transporte Proteico
6.
Cells ; 10(12)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34944035

RESUMO

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.


Assuntos
Envelhecimento/metabolismo , Doenças Cardiovasculares/metabolismo , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Membranas Mitocondriais/metabolismo , Transporte Proteico
7.
Chimia (Aarau) ; 75(12): 1026-1030, 2021 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-34920772

RESUMO

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.


Assuntos
Lipídeos , Proteínas , Membrana Celular , Transporte Proteico
8.
Cells ; 10(12)2021 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-34944018

RESUMO

Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus Alphavirus within the family Togaviridae. Humans infected with MAYV often develop chronic and debilitating arthralgia and myalgia. The virus is primarily maintained via a sylvatic cycle, but it has the potential to adapt to urban settings, which could lead to large outbreaks. The interferon (IFN) system is a critical antiviral response that limits replication and pathogenesis of many different RNA viruses, including alphaviruses. Here, we investigated how MAYV infection affects the induction phase of the IFN response. Production of type I and III IFNs was efficiently suppressed during MAYV infection, and mapping revealed that expression of the viral non-structural protein 2 (nsP2) was sufficient for this process. Interactome analysis showed that nsP2 interacts with DNA-directed RNA polymerase II subunit A (Rpb1) and transcription initiation factor IIE subunit 2 (TFIIE2), which are host proteins required for RNA polymerase II-mediated transcription. Levels of these host proteins were reduced by nsP2 expression and during infection by MAYV and related alphaviruses, suggesting that nsP2-mediated inhibition of host cell transcription is an important aspect of how some alphaviruses block IFN induction. The findings from this study may prove useful in design of vaccines and antivirals, which are currently not available for protection against MAYV and infection by other alphaviruses.


Assuntos
Alphavirus/metabolismo , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Subunidades Proteicas/metabolismo , Fatores de Transcrição TFII/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Regulação para Baixo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Ligação Proteica , Transporte Proteico , RNA Polimerase II/metabolismo , Transcrição Genética
9.
Int J Mol Sci ; 22(24)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34948308

RESUMO

Aquaporin-5 (AQP5) is selectively expressed in the apical membrane of exocrine glands, such as salivary, sweat, and submucosal airway glands, and plays important roles in maintaining their secretory functions. Because AQP5 is not regulated by gating, localization on the plasma membrane is important for its water-permeable function. Ezrin is an ezrin-radixin-moesin family protein that serves as a crosslinker between the plasma membrane and actin cytoskeleton network. It plays important roles in translocation of various membrane proteins to mediate vesicle trafficking to the plasma membrane. In this study, we examined the effects of ezrin inhibition on membrane trafficking of AQP5. Ezrin inhibition selectively suppressed an ionomycin-induced increase in AQP5 translocation to the plasma membrane of mouse lung epithelial cells (MLE-12) without affecting the steady-state level of plasma membrane AQP5. Taken together, our data suggest that AQP5 translocates to the plasma membrane through at least two pathways and that ezrin is selectively involved in a stimulation-dependent pathway.


Assuntos
Aquaporina 5/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ionóforos/metabolismo , Transporte Proteico/fisiologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo
10.
Cell Mol Life Sci ; 79(1): 15, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34967918

RESUMO

Excessive activation of the ionotropic N-methyl-D-aspartate (NMDA) receptor has been shown to cause abnormally high levels of Ca2+ influx, thereby leading to excitotoxic neuronal death. In this study, exposure of mouse primary cortical neurons to NMDA resulted in the cleavage and activation of mammalian sterile 20-like kinase-1 (MST1), both of which were mediated by calpain 1. In vitro cleavage assay data indicated that calpain 1 cleaves out the autoinhibitory domain of MST1 to generate an active form of the kinase. Furthermore, calpain 1 mediated the cleavage and activation of wild-type MST1, but not of MST1 (G339A). Intriguingly, NMDA/calpain-induced MST1 activation promoted the nuclear translocation of the kinase and the phosphorylation of histone H2B in mouse cortical neurons, leading to excitotoxicity. Thus, we propose a previously unrecognized mechanism of MST1 activation associated with NMDA-induced excitotoxic neuronal death.


Assuntos
Córtex Cerebral/patologia , Fator de Crescimento de Hepatócito/metabolismo , N-Metilaspartato/toxicidade , Neurônios/patologia , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Camundongos Endogâmicos C57BL , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Especificidade por Substrato/efeitos dos fármacos
11.
J Phys Chem Lett ; 12(51): 12249-12255, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34928612

RESUMO

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory's refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.


Assuntos
Proteínas do Envelope de Coronavírus/metabolismo , Simulação de Dinâmica Molecular , SARS-CoV-2/fisiologia , Proteínas da Matriz Viral/metabolismo , COVID-19/patologia , COVID-19/virologia , Proteínas do Envelope de Coronavírus/química , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Multimerização Proteica , Transporte Proteico , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/química
12.
Front Cell Infect Microbiol ; 11: 771653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34746036

RESUMO

The A chains of ADP-ribosylating toxins exploit Hsp90 for translocation into the host cytosol. Here, we hypothesize that cis proline residues play a key role in toxin recognition by Hsp90. Our model is largely derived from studies on the unusual interplay between Hsp90 and the catalytic A1 subunit of cholera toxin (CTA1), including the recent identification of an RPPDEI-like binding motif for Hsp90 in CTA1 and several other bacterial toxins. Cis/trans proline isomerization is known to influence protein-protein interactions and protein structure/function, but it has not yet been proposed to affect Hsp90-toxin interactions. Our model thus provides a new framework to understand the molecular basis for Hsp90 chaperone function and Hsp90-driven toxin translocation.


Assuntos
Toxinas Bacterianas , Prolina , Toxinas Bacterianas/metabolismo , Toxina da Cólera/metabolismo , Proteínas de Choque Térmico HSP90 , Isomerismo , Transporte Proteico
13.
PLoS One ; 16(10): e0258497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34624068

RESUMO

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.


Assuntos
Proteínas Hedgehog , Cílios , Flagelos/metabolismo , Transporte Proteico , Receptor Smoothened
14.
PLoS One ; 16(10): e0258111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34597321

RESUMO

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Imageamento Tridimensional/métodos , Membranas Intracelulares/metabolismo , Microscopia Confocal/métodos , Transporte Biológico , Transporte Proteico
15.
Nat Commun ; 12(1): 5773, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599159

RESUMO

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.


Assuntos
Inflamação/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Lipopolissacarídeos/farmacologia , Proteômica , Algoritmos , Anti-Infecciosos/metabolismo , Anti-Inflamatórios/metabolismo , Apresentação do Antígeno , Autofagossomos/metabolismo , Teorema de Bayes , Pontos de Checagem do Ciclo Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Forma Celular , Humanos , Imunidade , Inflamação/patologia , Leucemia/imunologia , Ativação Linfocitária/imunologia , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico , Proteoma/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Células THP-1 , Fatores de Tempo , Vesículas Transportadoras/metabolismo , Regulação para Cima , Proteínas rho de Ligação ao GTP/metabolismo
16.
Nat Commun ; 12(1): 5989, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645818

RESUMO

Liquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.


Assuntos
Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Triterpenos Pentacíclicos/farmacologia , Proteína Sequestossoma-1/genética , Animais , Complexo IV da Cadeia de Transporte de Elétrons , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão , Reologia , Proteína Sequestossoma-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
17.
ACS Chem Neurosci ; 12(21): 3972-3984, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34652126

RESUMO

Disrupted cellular trafficking and transport processes are hallmarks of many neurodegenerative disorders (NDs). Recently, efforts have been made toward developing and implementing experimental platforms to identify small molecules that may help restore normative trafficking functions. There have been a number of successes in targeting endomembrane trafficking with the identification of compounds that restore cell viability through rescue of protein transport and trafficking. Here, we describe some of the experimental platforms implemented for small molecule screening efforts for rescue of trafficking defects in neurodegeneration. A survey of phenotypically active small molecules identified to date is provided, including a summary of medicinal chemistry efforts and insights into putative targets and mechanisms of action. In particular, emphasis is put on ligands that demonstrate activity in more than one model of neurodegeneration as retention of phenotypic activity across ND models suggests conservation of biological targets across NDs.


Assuntos
Endossomos , Doenças Neurodegenerativas , Transporte Biológico , Endossomos/metabolismo , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Transporte Proteico
18.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638548

RESUMO

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.


Assuntos
Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Multimerização Proteica/genética , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismo , Linhagem Celular Tumoral , Cisteína/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/genética , Lectinas Tipo C/biossíntese , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Fosforilação , Domínios Proteicos/genética , Transporte Proteico/genética , Receptores Mitogênicos/biossíntese , Transdução de Sinais/imunologia
19.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638549

RESUMO

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antifúngicos/farmacologia , Cicloeximida/farmacologia , Endocitose/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação/efeitos dos fármacos
20.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638572

RESUMO

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.


Assuntos
Sinalização do Cálcio/genética , Expressão Gênica/genética , Neurônios Motores/fisiologia , Linhagem Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/fisiologia , Éxons/genética , Fibroblastos/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/fisiologia , Células HEK293 , Humanos , Atrofia Muscular Espinal/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Splicing de RNA/genética , RNA Mensageiro/genética , Proteínas do Complexo SMN/genética
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