RESUMEN
Peroxisome de novo biogenesis requires yet unidentified mitochondrial proteins. We report that the outer mitochondrial membrane (OMM)-associated E3 Ub ligase MARCH5 is vital for generating mitochondria-derived pre-peroxisomes. MARCH5 knockout results in accumulation of immature peroxisomes and lower expression of various peroxisomal proteins. Upon fatty acid-induced peroxisomal biogenesis, MARCH5 redistributes to newly formed peroxisomes; the peroxisomal biogenesis under these conditions is inhibited in MARCH5 knockout cells. MARCH5 activity-deficient mutants are stalled on peroxisomes and induce accumulation of peroxisomes containing high levels of the OMM protein Tom20 (mitochondria-derived pre-peroxisomes). Furthermore, depletion of peroxisome biogenesis factor Pex14 leads to the formation of MARCH5- and Tom20-positive peroxisomes, while no peroxisomes are detected in Pex14/MARCH5 dko cells. Reexpression of WT, but not MARCH5 mutants, restores Tom20-positive pre-peroxisomes in Pex14/MARCH5 dko cells. Thus, MARCH5 acts upstream of Pex14 in mitochondrial steps of peroxisome biogenesis. Our data validate the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process. Summary: The authors found that mitochondrial E3 Ub ligase MARCH5 controls the formation of mitochondria-derived pre-peroxisomes. The data support the hybrid, mitochondria-dependent model of peroxisome biogenesis and reveal that MARCH5 is an essential mitochondrial protein in this process.
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Among other mechanisms, mitochondrial membrane dynamics including mitochondrial fission and fusion, and the activity of the ubiquitin (Ub)-proteasome system (UPS) both are critical for maintaining mitochondrial function. To advance our knowledge of the role of mitochondrial fission, the UPS, and how they coordinatively affect mitochondrial response to proteotoxicity, we analyzed mitochondrial ubiquitination and mitochondria-specific autophagy (mitophagy) in E3 Ub ligase PRKN/parkin-expressing and -deficient cells. Through imaging, biochemical, and genetic analyses, we found that in a model of acute reduction of mitochondrial translation fidelity (MTF) some population of mitochondria within a single cell are enriched, while some showed reduced levels of CYCS (cytochrome c, somatic) and CPOX (coproporphyrinogen oxidase) proteins, both located in the intermembrane space (IMS); henceforth called "mosaic distribution". Formation of mosaic mitochondria requires mitochondrial fission and active mitochondrial translation. In cell lines deficient in PRKN activity, this process is followed by severing the outer mitochondrial membrane (OMM) and ubiquitination of the inner mitochondrial membrane (IMM) proteins (including TRAP1 and CPOX), recruitment of autophagy receptors, and formation of mito-autophagosomes. In contrast, in PRKN-expressing cells, mitochondria with high CYCS and CPOX levels are preferentially targeted by PRKN, leading to OMM ubiquitination and canonical PRKN-PINK1-mediated autophagy.
Asunto(s)
Autofagia , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.
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Autofagia , Dinaminas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Mitofagia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Citocromos c/metabolismo , Dinaminas/genética , Células HeLa , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Dinámicas Mitocondriales , Proteínas Mitocondriales/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Small ubiquitin-like modifier (SUMO) is a widespread regulatory mechanism of post-translational modification (PTM) that induces rapid and reversible changes in protein function and stability. Using SUMO conjugase Ubc9-overexpressing or knock-down cells in Parkinson's disease (PD) models, we demonstrate that SUMOylation protects dopaminergic cells against MPP+ or preformed fibrils (PFFs) of α-synuclein (α-syn)-induced toxicities in cell viability and cytotoxicity assays. In the mechanism of protection, Ubc9 overexpression significantly suppressed the MPP+ or PFF-induced reactive oxygen species (ROS) generation, while Ubc9-RNAi enhanced the toxicity-induced ROS production. Further, PFF-mediated protein aggregation was exacerbated by Ubc9-RNAi in thioflavin T staining, compared with NC1 controls. In cycloheximide (Chx)-based protein stability assays, higher protein level of α-syn was identified in Ubc9-enhanced green fluorescent protein (EGFP) than in EGFP cells. Since there was no difference in endogenous mRNA levels of α-syn between Ubc9 and EGFP cells in quantitative real-time PCR (qRT-PCR), we assessed the mechanisms of SUMO-mediated delayed α-syn degradation via MG132, proteasomal inhibitor, and PMA, lysosomal degradation inducer. Ubc9-mediated SUMOylated α-syn avoided PMA-induced lysosomal degradation because of its high solubility. Our results suggest that Ubc9 enhances the levels of SUMO1 and ubiquitin on α-syn and interrupts SUMO1 removal from α-syn. In immunohistochemistry, dopaminergic axon tips in the striatum and cell bodies in the substantia nigra from Ubc9-overexpressing transgenic mice were protected from MPTP toxicities compared with wild-type (WT) siblings. Our results support that SUMOylation can be a regulatory target to protect dopaminergic neurons from oxidative stress and protein aggregation, with the implication that high levels of SUMOylation in dopaminergic neurons can prevent the pathologic progression of PD.
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Enfermedad de Parkinson , Enzimas Ubiquitina-Conjugadoras , alfa-Sinucleína , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Ratones , Ratones Transgénicos , Ubiquitina , alfa-Sinucleína/genética , gamma-Glutamil HidrolasaRESUMEN
The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson's disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.
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BACKGROUND: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. METHODS: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). OUTCOMES: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. INTERPRETATION: We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.
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Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT(2)) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT(2) physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT(2), whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT(1) and with overexpressed VMAT(2). GST pull-down assays further identified three cytosolic domains of VMAT(2) involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT(2). Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT(2)-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT(2)/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT(2)-mediated transport into vesicles.
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Dopamina/biosíntesis , Dopamina/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Descarboxilasas de Aminoácido-L-Aromático/inmunología , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Transporte Biológico , Encéfalo/citología , Encéfalo/enzimología , Citosol/enzimología , Citosol/metabolismo , Humanos , Inmunoprecipitación , Masculino , Células PC12 , Estructura Terciaria de Proteína , Ratas , Vesículas Sinápticas/enzimología , Tirosina 3-Monooxigenasa/inmunología , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/química , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/fisiología , Islotes Pancreáticos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Receptores de Droga/metabolismo , Ubiquitina/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Animales , Células COS , Chlorocebus aethiops , Expresión Génica , Mutación , Canales de Potasio/genética , Canales de Potasio de Rectificación Interna/genética , Transporte de Proteínas , Receptores de Droga/genética , Receptores de SulfonilureasRESUMEN
The pancreatic ATP-sensitive potassium (K(ATP)) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to beta-cell membrane potential. Sulfonylureas inhibit K(ATP) channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K(ATP) channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K(ATP) channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K(ATP) channel trafficking mutations.
Asunto(s)
Adenosina Trifosfato/farmacología , Mutación , Canales de Potasio/metabolismo , Compuestos de Sulfonilurea/farmacología , Animales , Células COS , Gliburida/farmacología , Humanos , Transporte Iónico , Mediciones Luminiscentes , Técnicas de Placa-ClampRESUMEN
Mutations in the sulfonylurea receptor 1 (SUR1), a subunit of ATP-sensitive potassium (K(ATP)) channels, cause familial hyperinsulinism. One such mutation, deletion of phenylalanine 1388 (DeltaPhe-1388), leads to defects in both trafficking and MgADP response of K(ATP) channels. Here we investigated the biochemical features of Phe-1388 that control the proper trafficking and function of K(ATP) channels by substituting the residue with all other 19 amino acids. Whereas surface expression is largely dependent on hydrophobicity, channel response to MgADP is governed by multiple factors and involves the detailed architecture of the amino acid side chain. Thus, structural features in SUR1 required for proper channel function are distinct from those required for correct protein trafficking. Remarkably, replacing Phe-1388 by leucine profoundly alters the physiological and pharmacological properties of the channel. The F1388L-SUR1 channel has increased sensitivity to MgADP and metabolic inhibition, decreased sensitivity to glibenclamide, and responds to both diazoxide and pinacidil. Because this conservative amino acid substitution occurs in the SUR2A and SUR2B isoforms, the mutation provides a mechanism by which functional diversities in K(ATP) channels are generated.
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Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/farmacología , Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Adenosina Difosfato/metabolismo , Animales , Antiarrítmicos/farmacología , Western Blotting , Células COS , Cricetinae , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Gliburida/farmacología , Immunoblotting , Mediciones Luminiscentes , Magnesio/metabolismo , Ratones , Mutación , Técnicas de Placa-Clamp , Pinacidilo/farmacología , Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Receptores de Droga/química , Rubidio/farmacología , Receptores de Sulfonilureas , Factores de Tiempo , Transfección , Vasodilatadores/farmacologíaRESUMEN
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) is a potent and selective antagonist for the CB1 cannabinoid receptor. Using the AM1 molecular orbital method, conformational analysis of 1 around the pyrazole C3 substituent identified four distinct conformations designated Tg, Ts, Cg, and Cs. The energetic stability of these conformers followed the order Tg > Cg > Ts > Cs for the neutral (unprotonated) form of 1 and Ts > Tg > Cs > Cg for its piperidine N-protonated form. Unified pharmacophore models for the CB1 receptor ligands were developed by incorporating the protonated form of 1 into the superimposition model for the cannabinoid agonists 4-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]perhydro-2alpha,6beta-dihydroxynaphthalene (CP55244; 2) and the protonated form of (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2; 3) reported previously (Shim et al. In Rational Drug Design Symposium Series; Parrill, A. L., Reddy, M. R., Eds.; American Chemical Society: Washington, DC, 1999; pp 165-184). Values of K(i) for 1 and a series of 31 structural analogues were determined from radioligand binding analyses by competitive displacement of [3H]CP55940 from cannabinoid receptors in a rat brain membrane preparation. Comparative molecular field analysis (CoMFA) was employed to construct three-dimensional (3D)-quantitative structure-activity relationship (QSAR) models for this data set as unprotonated species assuming the Tg, Cg, and Ts conformers and for the protonated species assuming the Ts, Tg, and Cs conformers. Values of the conventional r2 and cross-validated r2 (r(cv)2) associated with these CoMFA models exceeded the threshold for statistical robustness (r2 > or = 0.90) and internal predictive ability (r(cv)2 > or = 0.50) in each of these six cases except for the protonated species assuming the Tg conformer (i.e., r2 = 0.97; r(cv)2 = 0.36). Results from conformational analyses, superimposition models, and 3D-QSAR models suggest that the N1 aromatic ring moiety of 1 dominates the steric binding interaction with the receptor in much the same way as does the C3 alkyl side chain of cannabinoid agonists and the C3 aroyl ring of the aminoalkylindole agonists. We also determined that several of the conformers considered in this study possess the proper spatial orientation and distinct electrostatic character to bind to the CB1 receptor. We propose that the unique region in space occupied by the C5 aromatic ring of 1 might contribute to conferring antagonist activity. We further propose that the pyrazole C3 substituent of 1 might contribute to conferring either neutral antagonist or inverse agonist activity, depending upon the interaction with the receptor.