RESUMEN
To propagate within a eukaryotic cell, pathogenic bacteria hijack and remodulate host cell functions. The Gram-negative obligate intracellular Chlamydiaceae, which pose a serious threat to human and animal health, attach to host cells and inject effector proteins that reprogram host cell machineries. Members of the conserved chlamydial TarP family have been characterized as major, early effectors that bind to and remodel the host actin cytoskeleton. We now describe a new function for the Chlamydia pneumoniae TarP member CPn0572, namely the ability to bind and alter the microtubule cytoskeleton. Thus, CPn0572 is unique in being the only prokaryotic protein that directly modulates both dynamic cytoskeletons of a eukaryotic cell. Ectopically expressed GFP-CPn0572 associates in a dose-independent manner with either cytoskeleton singly or simultaneously. In vitro, CPn0572 binds directly to microtubules. Expression of a microtubule-only CPn0572 variant resulted in the formation of an aberrantly thick, stabilized microtubule network. Intriguingly, during infection, secreted CPn0572 also co-localized with altered microtubules, suggesting that this protein also affects microtubule dynamics during infection. Our analysis points to a crosstalk between actin and microtubule cytoskeletons via chlamydial CPn0572.
RESUMEN
Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to C. pneumoniae infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating C. pneumoniae proteins, a screen in the model yeast Schizosaccharomyces pombe was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by C. pneumoniae EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced C. pneumoniae genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection.
Asunto(s)
Chlamydophila pneumoniae , Schizosaccharomyces , Humanos , Chlamydophila pneumoniae/metabolismo , Saccharomyces cerevisiae/metabolismo , Chlamydia trachomatis/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Insulin stimulates the exocytic translocation of specialized vesicles in adipocytes, which inserts GLUT4 glucose transporters into the plasma membrane to enhance glucose uptake. Previous results support a model in which TUG (Tether containing a UBX domain for GLUT4) proteins trap these GLUT4 storage vesicles at the Golgi matrix and in which insulin triggers endoproteolytic cleavage of TUG to translocate GLUT4. Here, we identify the muscle splice form of Usp25 (Usp25m) as a protease required for insulin-stimulated TUG cleavage and GLUT4 translocation in adipocytes. Usp25m is expressed in adipocytes, binds TUG and GLUT4, dissociates from TUG-bound vesicles after insulin addition, and colocalizes with TUG and insulin-responsive cargoes in unstimulated cells. Previous results show that TUG proteolysis generates the ubiquitin-like protein, TUGUL (for TUGubiquitin-like). We now show that TUGUL modifies the kinesin motor protein, KIF5B, and that TUG proteolysis is required to load GLUT4 onto these motors. Insulin stimulates TUG proteolytic processing independently of phosphatidylinositol 3-kinase. In nonadipocytes, TUG cleavage can be reconstituted by transfection of Usp25m, but not the related Usp25a isoform, together with other proteins present on GLUT4 vesicles. In rodents with diet-induced insulin resistance, TUG proteolysis and Usp25m protein abundance are reduced in adipose tissue. These effects occur soon after dietary manipulation, prior to the attenuation of insulin signaling to Akt. Together with previous data, these results support a model whereby insulin acts through Usp25m to mediate TUG cleavage, which liberates GLUT4 storage vesicles from the Golgi matrix and activates their microtubule-based movement to the plasma membrane. This TUG proteolytic pathway for insulin action is independent of Akt and is impaired by nutritional excess.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Cinesinas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Células Cultivadas , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Hipoglucemiantes/farmacología , Péptidos y Proteínas de Señalización Intracelular , Cinesinas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora , Transporte de Proteínas , Proteolisis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Ubiquitina Tiolesterasa/genéticaRESUMEN
Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.
Asunto(s)
Núcleo Celular/metabolismo , ARN Helicasas DEAD-box , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Nucleótidos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Conformación Proteica , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.
Asunto(s)
Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Hidrólisis , Mutación , Proteínas de Complejo Poro Nuclear/metabolismo , Fenotipo , Unión Proteica/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/fisiología , Cistinil Aminopeptidasa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Sirtuina 2/metabolismo , Células 3T3-L1 , Acetilación , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Cistinil Aminopeptidasa/genética , Citoplasma/metabolismo , Citometría de Flujo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 2/genéticaRESUMEN
In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.
Asunto(s)
Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Vasopresinas/metabolismo , Células 3T3-L1 , Animales , Transporte Biológico , Cistinil Aminopeptidasa/metabolismo , Exocitosis , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BLRESUMEN
Regulation of nuclear mRNA export is critical for proper eukaryotic gene expression. A key step in this process is the directional translocation of mRNA-ribonucleoprotein particles (mRNPs) through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. Our previous studies in Saccharomyces cerevisiae defined an in vivo role for inositol hexakisphosphate (InsP6) and NPC-associated Gle1 in mRNA export. Here, we show that Gle1 and InsP6 act together to stimulate the RNA-dependent ATPase activity of the essential DEAD-box protein Dbp5. Overexpression of DBP5 specifically suppressed mRNA export and growth defects of an ipk1 nup42 mutant defective in InsP6 production and Gle1 localization. In vitro kinetic analysis showed that InsP6 significantly increased Dbp5 ATPase activity in a Gle1-dependent manner and lowered the effective RNA concentration for half-maximal ATPase activity. Gle1 alone had minimal effects. Maximal InsP6 binding required both Dbp5 and Gle1. It has been suggested that Dbp5 requires unidentified cofactors. We now propose that Dbp5 activation at NPCs requires Gle1 and InsP6. This would facilitate spatial control of the remodelling of mRNP protein composition during directional transport and provide energy to power transport cycles.
Asunto(s)
Proteínas Portadoras/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ácido Fítico/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Sitios de Unión/fisiología , Proteínas Portadoras/genética , ARN Helicasas DEAD-box , Activación Enzimática/fisiología , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear , Proteínas de Transporte Nucleocitoplasmático/genética , ARN Helicasas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K family controls intracellular IP8 levels, the highest phosphorylated form of IPPs present in yeasts, as it has both inositol kinase and pyrophosphatase activities. Previous studies have shown that the fission yeast S. pombe Vip1/PPIP5K family member Asp1 impacts chromosome transmission fidelity via the modulation of spindle function. We now demonstrate that an IP8 analogue is targeted by endogenous Asp1 and that cellular IP8 is subject to cell cycle control. Mitotic entry requires Asp1 kinase function and IP8 levels are increased at the G2/M transition. In addition, the kinetochore, the conductor of chromosome segregation that is assembled on chromosomes is modulated by IP8. Members of the yeast CCAN kinetochore-subcomplex such as Mal2/CENP-O localize to the kinetochore depending on the intracellular IP8-level: higher than wild-type IP8 levels reduce Mal2 kinetochore targeting, while a reduction in IP8 has the opposite effect. As our perturbations of the inositol polyphosphate and IPP pathways demonstrate that kinetochore architecture depends solely on IP8 and not on other IPPs, we conclude that chromosome transmission fidelity is controlled by IP8 via an interplay between entry into mitosis, kinetochore architecture, and spindle dynamics.
RESUMEN
The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP(6)) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP(6) are also required for proper translation termination. However, the specificity of the IP(6)-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP(6) and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP(6) binding and show that these residues are needed for IP(6)-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP(6) for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP(6)-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP(6). We conclude that Gle1 specifically binds IP(6) and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Ácido Fítico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , ARN Helicasas DEAD-box/metabolismo , Glutatión Transferasa/metabolismo , Inositol/metabolismo , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Chlamydia pneumoniae is one of the two major species of the Chlamydiaceae family that have a profound effect on human health. C. pneumoniae is linked to a number of severe acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and infection by the pathogen might play a role in lung cancer. Following adhesion, Chlamydiae secrete effector proteins into the host cytoplasm that modulate the actin cytoskeleton facilitating internalization and infection. Members of the conserved TarP protein family comprise such effector proteins that polymerize actin, and in the case of the C. trachomatis TarP protein, has been shown to play a critical role in pathogenesis. In a previous study, we demonstrated that, upon bacterial invasion, the C. pneumoniae TarP family member CPn0572 is secreted into the host cytoplasm and recruits and associates with actin via an actin-binding domain conserved in TarP proteins. We have now extended our analysis of CPn0572 and found that the CPn0572 actin binding and modulating capability is more complex. With the help of the fission yeast system, a second actin modulating domain was identified independent of the actin binding domain. Microscopic analysis of HEp-2 cells expressing different CPn0572 deletion variants mapped this domain to the C-terminal part of the protein as CPn0572536-755 binds F-actin in vitro and colocalizes with aberrantly thickened actin cables in vivo. Finally, microscopic and bioinformatic analysis revealed the existence of a vinculin binding sequence in CPn0572. Our findings contribute to the understanding of the function of the TarP family and underscore the existence of several actin binding domains and a vinculin binding site for host actin modulation.
Asunto(s)
Proteínas Bacterianas/fisiología , Chlamydophila pneumoniae/patogenicidad , Vinculina/metabolismo , Actinas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/metabolismo , Biología Computacional , Citoesqueleto/metabolismo , Humanos , Dominios Proteicos , Análisis de Secuencia de ProteínaRESUMEN
Translocation of messenger RNAs through the nuclear pore complex (NPC) requires coordinated physical interactions between stable NPC components, shuttling transport factors, and mRNA-binding proteins. In budding yeast (y) and human (h) cells, Gle1 is an essential mRNA export factor. Nucleocytoplasmic shuttling of hGle1 is required for mRNA export; however, the mechanism by which hGle1 associates with the NPC is unknown. We have previously shown that the interaction of hGle1 with the nucleoporin hNup155 is necessary but not sufficient for targeting hGle1 to NPCs. Here, we report that the unique C-terminal 43 amino acid region of the hGle1B isoform mediates binding to the C-terminal non-FG region of the nucleoporin hCG1/NPL1. Moreover, hNup155, hGle1B, and hCG1 formed a heterotrimeric complex in vitro. This suggested that these two nucleoporins were required for the NPC localization of hGle1. Using an siRNA-based approach, decreased levels of hCG1 resulted in hGle1 accumulation in cytoplasmic foci. This was coincident with inhibition of heat shock-induced production of Hsp70 protein and export of the Hsp70 mRNA in HeLa cells. Because this closely parallels the role of the hCG1 orthologue yNup42/Rip1, we speculate that hGle1-hCG1 function in the mRNA export mechanism is highly conserved.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , Transporte Biológico/genética , Proteínas Portadoras/genética , Secuencia Conservada , Genes Reporteros , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Humanos , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/deficiencia , Proteínas de Transporte Nucleocitoplasmático/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The plasma membrane delineates the cell and mediates its communication and material exchange with the environment. Many processes of the plasma membrane occur through interactions of proteins with phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2), which is highly enriched in this membrane and is a key determinant of its identity. Eisosomes function in lateral organization of the plasma membrane, but the molecular function of their major protein subunits, the BAR domain-containing proteins Pil1 and Lsp1, is poorly understood. Here we show that eisosomes interact with the PI(4,5)P2 phosphatase Inp51/Sjl1, thereby recruiting it to the plasma membrane. Pil1 is essential for plasma membrane localization and function of Inp51 but not for the homologous phosphatidylinositol bisphosphate phosphatases Inp52/Sjl2 and Inp53/Sjl3. Consistent with this, absence of Pil1 increases total and available PI(4,5)P2 levels at the plasma membrane. On the basis of these findings, we propose a model in which the eisosomes function in maintaining PI(4,5)P2 levels by Inp51/Sjl1 recruitment.
Asunto(s)
Orgánulos/enzimología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular , Orgánulos/fisiología , Fosfoproteínas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.
Asunto(s)
Dermis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Tiadiazoles/farmacología , Triazoles/farmacología , Células Cultivadas , Dermis/citología , Dermis/enzimología , Ensayo de Cambio de Movilidad Electroforética , Inhibidores Enzimáticos/química , Fibroblastos/citología , Fibroblastos/enzimología , Polarización de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Inositol Polifosfato 5-Fosfatasas , Estructura Molecular , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Colorantes de Rosanilina , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Tiadiazoles/química , Triazoles/químicaRESUMEN
Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for (~)35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and ß isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
Asunto(s)
Autofagia/fisiología , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Animales , Línea Celular , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Técnicas de Inactivación de Genes , Humanos , Espacio Intracelular/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Fagosomas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica , Proteína Fosfatasa 2C , ProteolisisRESUMEN
Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.
Asunto(s)
Interleucina-12/antagonistas & inhibidores , Interleucina-23/antagonistas & inhibidores , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal/efectos de los fármacos , Receptores Toll-Like/metabolismo , Triazinas/farmacología , Animales , Línea Celular , Citocinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazonas , Ratones , Morfolinas/metabolismo , Unión Proteica , Pirimidinas , Especificidad por Sustrato , Triazinas/metabolismoRESUMEN
Highly phosphorylated, soluble inositides are an emerging family of potential eukaryotic second messengers. The mechanisms for generating an outstanding diversity of mono- and pyrophosphorylated inositides have been recently elucidated and require a series of conserved lipases, kinases, and phosphatases. With several of the inositol kinases and the phospholipase C having access to the nucleus, roles for inositides in nuclear functions have been suggested. In support of this hypothesis, multiple studies have revealed the protein machines that are modulated by these inositides and found specific roles in nuclear physiology. In this paper, we review a novel paradigm for regulating gene expression at distinct steps by different inositide isomers. We discuss discoveries showing inositol polyphosphate regulation of gene expression at the level of transcription, chromatin remodeling, messenger ribonucleic acid (mRNA) editing, and mRNA export. Recent structural studies of inositol polyphosphate-binding proteins suggest the inositides modulate protein function as essential structural cofactors, triggers for allosteric or induced fit structural changes, and direct antagonistic competitors for other inositide ligands. We propose that the cell orchestrates the localized production of soluble inositol polyphosphates and inositol pyrophosphates to direct decisive and rapid signaling events. These insights also illustrate how extracellular stimuli might faithfully trigger the correct synchrony between gene expression steps and coordinate nuclear responses to changes in cellular environments.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Fosfatos de Inositol/fisiología , Animales , HumanosRESUMEN
Nuclear export of mRNA is mediated by interactions between soluble factors and nuclear pore complex (NPC) proteins. In Saccharomyces cerevisiae, Nab2 is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm. The mechanism for trafficking of Nab2-bound mRNA through the NPC has not been defined. Gle1 is also required for mRNA export, and Gle1 interactions with NPC proteins, the RNA helicase Dbp5, and Gfd1 have been reported. Here we report that Nab2, Gfd1, and Gle1 associate in a complex. By using immobilized recombinant Gfd1, Nab2 was isolated from total yeast lysate. A similar biochemical assay with immobilized recombinant Nab2 resulted in coisolation of Gfd1 and Gle1. A Nab2-Gfd1 complex was also identified by coimmunoprecipitation from yeast lysates. In vitro binding assays with recombinant proteins revealed a direct association between Nab2 and Gfd1, and two-hybrid assays delineated Gfd1 binding to the N-terminal Nab2 domain. This N-terminal Nab2 domain is distinct from its RNA binding domains suggesting Nab2 could bind Gfd1 and RNA simultaneously. As Nab2 export was blocked in a gle1 mutant at the restrictive temperature, we propose a model wherein Gfd1 serves as a bridging factor between Gle1 and Nab2-bound mRNA during export.