RESUMO
For the past several years, fundamental research on Sigma-1R (S1R) protein has unveiled its necessity for maintaining proper cellular homeostasis through modulation of calcium and lipid exchange between the endoplasmic reticulum (ER) and mitochondria, ER-stress response, and many other mechanisms. Most of these processes, such as ER-stress response and autophagy, have been associated with neuroprotective roles. In fact, improving these mechanisms using S1R agonists was beneficial in several brain disorders including neurodegenerative diseases. In this review, we will examine S1R subcellular localization and describe S1R-associated biological activity within these specific compartments, i.e., the Mitochondrion-Associated ER Membrane (MAM), ER-Lipid Droplet (ER-LD) interface, ER-Plasma Membreane (ER-PM) interface, and the Nuclear Envelope (NE). We also discussed how the dysregulation of these pathways contributes to neurodegenerative diseases, while highlighting the cellular mechanisms and key binding partners engaged in these processes.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Doenças Neurodegenerativas , Neuroproteção , Receptores sigma , Humanos , Autofagia/genética , Autofagia/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Neuroproteção/genética , Neuroproteção/fisiologia , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Receptores sigma/genética , Receptores sigma/metabolismo , Receptor Sigma-1RESUMO
Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.
Assuntos
Autofagia/genética , Efeito Fundador , Genes Recessivos , Leucoencefalopatias/genética , Mutação , Proteínas de Transporte Vesicular/genética , Adulto , Sequência de Aminoácidos , Animais , Morte Celular/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/química , Adulto JovemRESUMO
We and others have shown that trafficking of G-protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Here, we show by overexpression and depletion studies that HACE1, a HECT-domain-containing ubiquitin ligase, promotes the recycling of the ß2-adrenergic receptor (ß2AR), a prototypical G-protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the ß2AR in conjunction with HACE1 triggered ubiquitylation of Rab11a, as observed by western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitylated on Lys145. A Rab11a-K145R mutant failed to undergo ß2AR-HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the ß2AR. Rab11a, but not Rab11a-K145R, was activated by ß2AR-HACE1, indicating that ubiquitylation of Lys145 is involved in activation of Rab11a. Co-expression of ß2AR-HACE1 also potentiated ubiquitylation of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases through their ubiquitylation, with associated functional effects demonstrated on Rab11a. This suggests a new pathway whereby a cargo protein, such as a G-protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitylation and activation of a Rab GTPase.
Assuntos
Receptores Adrenérgicos beta 2/genética , Ubiquitina-Proteína Ligases/genética , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Arginina/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Transporte Proteico , Receptores Adrenérgicos beta 2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The vertebrate planar cell polarity (PCP) pathway shares molecular components with the ß-catenin-mediated canonical Wnt pathway but acts through membrane complexes containing Vang or Frizzled to orient neighboring cells coordinately. The molecular interactions underlying the action of Vang in PCP signaling and specification, however, are yet to be delineated. Here, we report the identification of Rack1 as an interacting protein of a vertebrate Vang protein, Vangl2. We demonstrate that Rack1 is required in zebrafish for PCP-regulated processes, including oriented cell division, cellular polarization, and convergent extension during gastrulation. We further show that the knockdown of Rack1 affects membrane localization of Vangl2 and that the Vangl2-interacting domain of Rack1 has a dominant-negative effect on Vangl2 localization and gastrulation. Moreover, Rack1 antagonizes canonical Wnt signaling. Together, our data suggest that Rack1 regulates the localization of an essential PCP protein and acts as a molecular switch to promote PCP signaling.
Assuntos
Polaridade Celular/fisiologia , Gástrula/metabolismo , Gastrulação/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Divisão Celular/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Gástrula/citologia , Proteínas de Membrana/genética , Camundongos , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Proteínas Wnt/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the ß(2)-adrenergic receptor (ß(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that ß(2)AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L(339)L(340) motif in the ß(2)AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the ß(2)AR, which requires the interaction of RGGTA with the ß(2)AR L(339)L(340) motif. Furthermore, the ß(2)AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the ß(2)AR was dependent on the RGGTA-interacting L(339)L(340) motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted ß(2)AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.
Assuntos
Alquil e Aril Transferases/metabolismo , Prenilação de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Células HEK293 , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Especificidade por Substrato , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Dysfunctional autophagy is implicated in Alzheimer's Disease (AD) pathogenesis. The alterations in the expression of many autophagy related genes (ATGs) have been reported in AD brains; however, the disparity of the changes confounds the role of autophagy in AD. METHODS: To further understand the autophagy alteration in AD brains, we analyzed transcriptomic (RNAseq) datasets of several brain regions (BA10, BA22, BA36 and BA44 in 223 patients compared to 59 healthy controls) and measured the expression of 130 ATGs. We used autophagy-deficient mouse models to assess the impact of the identified ATGs depletion on memory, autophagic activity and amyloid-ß (Aß) production. RESULTS: We observed significant downregulation of multiple components of two autophagy kinase complexes BECN1-PIK3C3 and ULK1/2-FIP200 specifically in the parahippocampal gyrus (BA36). Most importantly, we demonstrated that deletion of NRBF2, a component of the BECN1-PIK3C3 complex, which also associates with ULK1/2-FIP200 complex, impairs memory in mice, alters long-term potentiation (LTP), reduces autophagy in mouse hippocampus, and promotes Aß accumulation. Furthermore, AAV-mediated NRBF2 overexpression in the hippocampus not only rescues the impaired autophagy and memory deficits in NRBF2-depleted mice, but also reduces ß-amyloid levels and improves memory in an AD mouse model. CONCLUSIONS: Our data not only implicates NRBF2 deficiency as a risk factor for cognitive impairment associated with AD, but also support the idea of NRBF2 as a potential therapeutic target for AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas Relacionadas à Autofagia/genética , Autofagia/fisiologia , Memória/fisiologia , Transativadores/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Transtornos da Memória/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismoRESUMO
Previously thought of as a nonselective digestion process, autophagy is now known to specifically degrade aggregated proteins and damaged cellular organelles through the action of autophagy receptors, which provides cellular quality control and maintains homeostasis. Autophagy receptors recognize and recruit specific cargoes to the autophagosome-lysosome pathway for degradation in ubiquitin-dependent and -independent manners, and their functions (in selective autophagy) are regulated by protein modifications, for example, phosphorylation and ubiquitination. Growing evidence has linked the genetic variants of autophagy receptors to neurodegenerative diseases and multiple experimental systems have validated their roles in modulating the disease process. Here, we review the recent advances in understanding the physiology and pathophysiology of autophagy receptors in selective autophagy, and discuss their potentials as therapeutic targets for neurodegenerative diseases.
Assuntos
Autofagia , Doenças Neurodegenerativas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , HumanosRESUMO
BACKGROUND: Autophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn't fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington's disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers. METHOD: We generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington's disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation. RESULTS: Here, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington's disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells. CONCLUSION: Our study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Doença de Huntington/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Microscopia de Fluorescência , FosforilaçãoRESUMO
Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Growing evidence suggests that mutations, aberrant expression, and altered post-translational modifications of Rab GTPases are associated with human diseases. However, their regulatory mechanisms and how they are connected to cargo proteins are still poorly understood. Accumulating data indicate that G protein-coupled receptors (GPCRs) directly associate with Rab GTPases and that these interactions dictate receptor trafficking. Yet, it remained unclear whether the receptors could regulate the targeting and activity of Rab GTPases in various cell compartments. It is only in recent years that experimental studies showed that GPCR signaling and interaction with Rab-associated regulatory proteins modulate the localization and activity of Rab GTPases. This research is revealing novel regulatory mechanisms of these small GTPases and should contribute to the progress in effective drug development. Recently published in the Journal of Cell Science, Lachance et al. present a novel role for ubiquitylation of Rab11a by a ß2AR/HACE1 complex in regulating Rab11a activity and ß2AR trafficking.