RESUMEN
IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.
Asunto(s)
Aciltransferasas , Retículo Endoplásmico , Glicoproteínas Hemaglutininas del Virus de la Influenza , Virus de la Influenza B , Lipoilación , Ácido Palmítico , Replicación Viral , Humanos , Aciltransferasas/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/química , Virus de la Influenza A/metabolismo , Virus de la Influenza B/química , Virus de la Influenza B/crecimiento & desarrollo , Virus de la Influenza B/metabolismo , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Lipoilación/genética , Mutación , Ácido Palmítico/metabolismoRESUMEN
Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D, and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore-induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post-translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 directly regulates protein lipoylation by binding the lipoyl synthase (LIAS) enzyme promoting its functional binding to the lipoyl carrier protein GCSH and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss of function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling established that FDX1 loss-of-function results in the induction of both compensatory metabolism-related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-function is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting its role in cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.
Asunto(s)
Ferredoxinas , Lipoilación , Sulfurtransferasas , Humanos , Ferredoxinas/genética , Ferredoxinas/metabolismo , Lipoilación/genética , Unión Proteica , Respiración de la Célula/genética , Proliferación Celular/genética , Metaboloma , Sulfurtransferasas/metabolismoRESUMEN
Palmitoylation is a post-translational modification occurring on cysteine residues, which process is catalyzed by a family of zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases. As a family member, ZDHHC9 plays a crucial role in varied malignancies by regulating protein stability via protein substrate palmitoylation. Based on the bioinformatic analysis of GEO gene microarray GSE75037 (|log2 fold change|> 1, P < 0.05), ZDHHC9 was defined as a significantly upregulated gene in lung adenocarcinoma (LUAD), which was also confirmed in our collected clinical specimens. It is necessary to explore the biological function of ZDHHC9 in LUAD cells. The follow-up functional experiments revealed that ZDHHC9 deficiency inhibited proliferation, migration, and invasion, while stimulated apoptosis in HCC827 cells. Besides, these malignant phenotypes could be accelerated by ZDHHC9 overexpression in A549. Moreover, we revealed that ZDHHC9 knockdown could promote PD-L1 protein degradation by reducing its palmitoylation level. The reduction of PD-L1 protein level could enhance anti-tumor immunity and inhibit the growth of LUAD cells. Therefore, our study uncovers the tumor-promoting role of ZDHHC9 in LUAD via regulating PD-L1 stability through palmitoylation, highlighting ZDHHC9 as a novel therapeutic target for LUAD.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Animales , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Lipoilación/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Procesamiento Proteico-PostraduccionalRESUMEN
The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ácido Fólico/metabolismo , Glicina/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Humanos , Lipoilación/genética , Proteínas Mitocondriales/metabolismo , Nucleótidos/metabolismoRESUMEN
Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. "Plastid-type" NFU1, NFU2, and NFU3 in Arabidopsis (Arabidopsis thaliana) play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here, we confirmed that NFU4 and NFU5 are targeted to the mitochondria. The proteins were constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of NFU4 and NFU5 proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes or aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth improved with elevated CO2 treatment. In addition, pyruvate, 2-oxoglutarate, and branched-chain amino acids accumulated in nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, most likely providing Fe-S clusters to lipoyl synthase.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Lipoilación/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , MutaciónRESUMEN
Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulated. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that mHTT palmitoylation decreases further with aging. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons.
Asunto(s)
Proteína Huntingtina/genética , Proteína Huntingtina/toxicidad , Lipoilación/efectos de los fármacos , Lipoilación/genética , Aciltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Cisteína/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , RatasRESUMEN
Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnß transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnß transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Inmunidad Innata , Macrófagos/inmunología , Sirtuinas/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , ARN Helicasas DEAD-box/genética , Células HEK293 , Humanos , Lipoilación/genética , Lipoilación/inmunología , Macrófagos/virología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Células RAW 264.7 , Sirtuinas/genética , Estomatitis Vesicular/genética , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
TITLE: PolDIP2, une protéine clé de la régulation du fonctionnement mitochondrial et du métabolisme cellulaire. ABSTRACT: Pour la sixième année, dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-sud, les étudiants du Master « Biologie Santé ¼ de l'université Paris-Saclay se sont confrontés à l'écriture scientifique. Ils ont sélectionné une quinzaine d'articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l'originalité de l'article étudié. Ils ont beaucoup apprécié cette initiation à l'écriture d'articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Trois de ces Nouvelles sont publiées dans ce numéro, les autres le seront dans des prochains numéros.
Asunto(s)
Metabolismo Energético/genética , Mitocondrias/fisiología , Proteínas Nucleares/fisiología , Animales , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Ciclo del Ácido Cítrico/genética , Humanos , Lipoilación/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity.
Asunto(s)
Inmunidad Celular/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/genética , Acilación/genética , Aciltransferasas/química , Aciltransferasas/genética , Membrana Celular/química , Membrana Celular/genética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Inmunidad Celular/inmunología , Lipoilación/genética , Mutación/genética , Procesamiento Proteico-Postraduccional/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/genética , Especificidad por Sustrato/genética , Linfocitos T/química , Proteína Tirosina Quinasa ZAP-70/químicaRESUMEN
Protein palmitoylation, in which C16 fatty acid chains are attached to cysteine residues via a reversible thioester linkage, is one of the most common lipid modifications and plays important roles in regulating protein stability, subcellular localization, membrane trafficking, interactions with effector proteins, enzymatic activity, and a variety of other cellular processes. Moreover, the unique reversibility of palmitoylation allows proteins to be rapidly shuttled between biological membranes and cytoplasmic substrates in a process usually controlled by a member of the DHHC family of protein palmitoyl transferases (PATs). Notably, mutations in PATs are closely related to a variety of human diseases, such as cancer, neurological disorders, and immune deficiency conditions. In addition to PATs, intracellular palmitoylation dynamics are also regulated by the interplay between distinct posttranslational modifications, including ubiquitination and phosphorylation. Understanding the specific mechanisms of palmitoylation may reveal novel potential therapeutic targets for many human diseases.
Asunto(s)
Aciltransferasas/metabolismo , Lipoilación/genética , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Animales , Metilación de ADN/fisiología , Humanos , Especificidad por Sustrato/fisiologíaAsunto(s)
Enfermedades Autoinmunes/genética , Inflamación/genética , Lipoilación/genética , Ácido Palmítico/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Sustitución de Aminoácidos/genética , Arginina/genética , Enfermedades Autoinmunes/metabolismo , Cisteína/genética , Humanos , Inflamación/metabolismo , Lipoilación/fisiología , Mutación Missense , SíndromeRESUMEN
Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.
In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than 'normal' cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Mioblastos/fisiología , Animales , Diferenciación Celular , Línea Celular , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Células HEK293 , Humanos , Lipoilación/genética , Ratones , Oxidación-ReducciónRESUMEN
Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by zDHHC5. Here, we show that PLM palmitoylation is facilitated by recruitment of the Na-pump α sub-unit to a specific site on zDHHC5 that contains a juxtamembrane amphipathic helix. Site-specific palmitoylation and GlcNAcylation of this helix increased binding between the Na-pump and zDHHC5, promoting PLM palmitoylation. In contrast, disruption of the zDHHC5-Na-pump interaction with a cell penetrating peptide reduced PLM palmitoylation. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease.
Asunto(s)
Acetiltransferasas/genética , Aciltransferasas/genética , Lipoilación/genética , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Animales , Membrana Celular/genética , Péptidos de Penetración Celular/genética , Humanos , Ratones , Fosforilación/genética , Procesamiento Proteico-Postraduccional/genética , Ratas , ATPasa Intercambiadora de Sodio-Potasio/genética , Especificidad por Sustrato/genéticaRESUMEN
Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K+ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.
Asunto(s)
Aciltransferasas/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Habituación Psicofisiológica/genética , Aprendizaje/fisiología , Lipoilación/genética , Lipoilación/fisiología , Proteínas del Tejido Nervioso/fisiología , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Canales de Potasio de la Superfamilia Shaker/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Terminales Presinápticos/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismoRESUMEN
Pancreatic cancer (PC) is one of the deadliest cancers worldwide. Cancer cells secrete excessive numbers of exosomes that play essential roles in tumorigenesis. Long non-coding RNAs (lncRNAs) are essential non-coding RNAs for cancer progression. However, the role of lncRNA plasmacytoma variant translocation 1 (PVT1) in exosome secretion of PC remains to be comprehensively investigated. Thus, nanoparticle tracking analysis and transmission electron microscopy were performed to determine exosome secretion. Confocal microscopy, western blots, real-time PCR, immunofluorescence, pull-down and RNA immunoprecipitation assays, and rescue experiments were applied to investigate the mechanism underlying the role of PVT1 in exosome secretion. The results showed that PVT1 was upregulated in PC cells, along with increased levels of YKT6 v-SNARE homolog (YKT6), ras-related protein Rab-7 (RAB7), and vesicle-associated membrane protein 3 (VAMP3). Also, PVT1 promoted the transportation of multivesicular bodies (MVBs) towards the plasma membrane. In addition, PVT1 promoted the docking of MVBs by altering RAB7 expression and localization. Moreover, PVT1 promoted the fusion of MVBs with the plasma membrane through regulating YKT6 and VAMP3 colocalization and the palmitoylation of YKT6. Taken together, the results suggest that PVT1 promoted exosome secretion of PC cells and thus, can expand the understanding of PVT1 in tumor biology.
Asunto(s)
Exosomas/metabolismo , Neoplasias Pancreáticas/patología , ARN Largo no Codificante/metabolismo , Microambiente Tumoral/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Exosomas/ultraestructura , Regulación Neoplásica de la Expresión Génica , Humanos , Lipoilación/genética , Microscopía Electrónica de Transmisión , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Neoplasias Pancreáticas/genética , Proteínas R-SNARE/genética , Regulación hacia Arriba , Proteína 3 de Membrana Asociada a Vesículas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
The phylum Apicomplexa includes a number of significant human pathogens like Toxoplasma gondii and Plasmodium species. These obligate intracellular parasites possess a membranous structure, the inner membrane complex (IMC), composed of flattened vesicles apposed to the plasma membrane. Numerous proteins associated with the IMC are anchored via a lipid post-translational modification termed palmitoylation. This acylation is catalysed by multi-membrane spanning protein S-acyl-transferases (PATs) containing a catalytic Asp-His-His-Cys (DHHC) motif, commonly referred to as DHHCs. Contrasting the redundancy observed in other organisms, several PATs are essential for T. gondii tachyzoite survival; 2 of them, TgDHHC2 and TgDHHC14 being IMC-resident. Disruption of either of these TgDHHCs results in a rapid collapse of the IMC in the developing daughter cells leading to dramatic morphological defects of the parasites while the impact on the other organelles is limited to their localisation but not to their biogenesis. The acyl-transferase activity of TgDHHC2 and TgDHHC14 is involved sequentially in the formation of the sub-compartments of the IMC. Investigation of proteins known to be palmitoylated and localised to these sub-compartments identified TgISP1/3 as well as TgIAP1/2 to lose their membrane association revealing them as likely substrates of TgDHHC2, while these proteins are not impacted by TgDHHC14 depletion.
Asunto(s)
Aciltransferasas/metabolismo , Membranas Intracelulares/fisiología , Lipoilación/genética , Biogénesis de Organelos , Toxoplasma/enzimología , Toxoplasma/fisiología , Acilación , Aciltransferasas/clasificación , Aciltransferasas/genética , Lipoilación/fisiología , Procesamiento Proteico-Postraduccional , Toxoplasma/genéticaRESUMEN
Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Hematopoyesis/genética , Lipoilación/genética , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Sustitución de Aminoácidos , Animales , Ácido Aspártico/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Glicina/genética , Neoplasias Hematológicas/metabolismo , Células Madre Hematopoyéticas/fisiología , Redes y Vías Metabólicas/genética , Ratones , Ratones Transgénicos , Ácido Palmítico/metabolismoRESUMEN
Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.
Asunto(s)
Colesterol/genética , Lipoilación/genética , Microdominios de Membrana/genética , Receptores de Lipoproteína/genética , Comunicación Celular/genética , Colesterol/metabolismo , Cisteína/química , Cisteína/genética , Células Epiteliales/metabolismo , Humanos , Uniones Intercelulares/genética , Proteína 2 con Dominio MARVEL , Microdominios de Membrana/química , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , Receptores de Lipoproteína/química , Uniones Estrechas/genética , Uniones Estrechas/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, must adapt to host-associated environments during infection by modulating gene expression. Small regulatory RNAs (sRNAs) are key regulators of bacterial gene expression, but their roles in Mtb are not well understood. Here, we address the expression and function of the Mtb sRNA Mcr11, which is associated with slow bacterial growth and chronic infections in mice. We found that stable expression of Mcr11 requires multiple factors specific to TB-complex bacteria, including the AbmR transcription factor. Bioinformatic analyses used to predict regulatory targets of Mcr11 identified 7-11 nucleotide regions with potential for direct base-pairing with Mcr11 immediately upstream of Rv3282, fadA3, and lipB. mcr11-dependent regulation of these genes was demonstrated using qRT-PCR and found to be responsive to the presence of fatty acids. Mutation of the putative Mcr11 base-pairing site upstream of lipB in a promoter reporter strain resulted in significant de-repression of lipB expression, similar to that observed in mcr11-deleted Mtb. These studies establish Mcr11's roles in regulating growth and central metabolism in Mtb. Our finding that multiple TB-complex-specific factors are required for production of stable Mcr11 also emphasizes the need to better understand mechanisms of sRNA expression and stability in TB.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Mycobacterium tuberculosis , ARN Pequeño no Traducido/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Biología Computacional , Genes Bacterianos , Lipoilación/genética , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as their natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here, we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site-codon 70-within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of representatives of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72, and C105, mutation of each cysteine individually impairs virus restriction, and a triple C71A-C72A-C105A mutant loses all restriction activity, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation.