RESUMEN
LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.
Asunto(s)
Quinasas Lim , Inhibidores de Proteínas Quinasas , Quinasas Lim/antagonistas & inhibidores , Quinasas Lim/metabolismo , Humanos , Relación Estructura-Actividad , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Estructura Molecular , Movimiento Celular/efectos de los fármacos , Modelos Moleculares , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Relación Dosis-Respuesta a Droga , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/síntesis químicaRESUMEN
Surface display co-opts yeast's innate ability to embellish its cell wall with mannoproteins, thus converting the yeast's outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoform's N-terminus, e.g., through a short GPI-independent protein anchor. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the ß-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Pared Celular , Genómica , GlucanosRESUMEN
LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of the LIM kinase family harbouring two LIM domains at their N-terminus and a kinase domain at their C-terminus [...].
Asunto(s)
Quinasas Lim , Quinasas Lim/genéticaRESUMEN
LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell.
Asunto(s)
Citoesqueleto , Quinasas Lim , Quinasas Lim/metabolismo , Fosforilación , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Diferenciación CelularRESUMEN
BACKGROUND: Engineered living materials (ELMs) combine living cells with non-living scaffolds to obtain life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs can be 3D-printed and are called bioinks, and their scaffolds are mostly hydrogel-based. One such scaffold is polymer Pluronic F127, a liquid at 4 °C but a biocompatible hydrogel at room temperature. In such thermally-reversible hydrogel, the microorganism-hydrogel interactions remain uncharacterized, making truly durable 3D-bioprinted ELMs elusive. METHODS: We demonstrate the methodology to assess cell-scaffold interactions by characterizing intact alive yeast cells in cross-linked F127-based hydrogels, using genetically encoded ratiometric biosensors to measure intracellular ATP and cytosolic pH at a single-cell level through confocal imaging. RESULTS: When embedded in hydrogel, cells were ATP-rich, in exponential or stationary phase, and assembled into microcolonies, which sometimes merged into larger superstructures. The hydrogels supported (micro)aerobic conditions and induced a nutrient gradient that limited microcolony size. External compounds could diffuse at least 2.7 mm into the hydrogels, although for optimal yeast growth bioprinted structures should be thinner than 0.6 mm. Moreover, the hydrogels could carry whole-cell copper biosensors, shielding them from contaminations and providing them with nutrients. CONCLUSIONS: F127-based hydrogels are promising scaffolds for 3D-bioprinted ELMs, supporting a heterogeneous cell population primarily shaped by nutrient availability.
RESUMEN
LIM Kinases are important actors in the regulation of cytoskeleton dynamics by controlling microtubule and actin filament turnover. The signaling pathways involving LIM kinases for actin filament remodeling are well established. They are downstream effectors of small G proteins of the Rho-GTPases family and have become promising targets for the treatment of several major diseases because of their position at the lower end of these signaling cascades. Cofilin, which depolymerizes actin filaments, is the best-known substrate of these enzymes. The phosphorylation of cofilin to its inactive form by LIM kinases avoids actin filament depolymerization. The balance between phosphorylated and non-phosphorylated cofilin is thought to play an important role in tumor cell invasion and metastasis. Since 2006, many small molecules have been developed for LIMK inhibition, and in this review article, we will discuss the structure-activity relationships of the few inhibitor families that have been tested in vivo on different pathological models.
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Actinas , Quinasas Lim , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Humanos , Quinasas Lim/metabolismoRESUMEN
While copper is an essential micronutrient and a technologically indispensable heavy metal, it is toxic at high concentrations, harming the environment and human health. Currently, copper is monitored with costly and low-throughput analytical techniques that do not evaluate bioavailability, a crucial parameter which can be measured only with living cells. We overcame these limitations by building upon yeast S. cerevisiae's native copper response and constructed a promising next-generation eukaryotic whole-cell copper biosensor. We combined a dual-reporter fluorescent system with an engineered CUP1 promoter and overexpressed Cup2 transactivator, constructing through four iterations a total of 16 variants of the biosensor, with the best one exhibiting a linear range of 10-8 to 10-3 M of bioavailable copper. The engineered variant distinguishes itself through superior specificity, detection limit, and linear range, compared to other currently reported eukaryotic and prokaryotic whole-cell copper biosensors. Moreover, the variant serves as a dual-sensing reporter for Cu2+ detection and cell viability, disregards non-bioavailable copper and other heavy metals, is relatively independent of the cell's physiological status, and was validated on real-world samples which contained interfering substances. Finally, by re-engineering the transactivator, we altered the system's sensitivity and growth rate while assessing the performance of Cup2 with heterologous activation domains. Thus, in addition to presenting the next-generation whole-cell copper biosensor, this work urges for an iterative design of eukaryotic biosensors and paves the way toward higher sensitivity through transactivator engineering.
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Técnicas Biosensibles , Metales Pesados , Técnicas Biosensibles/métodos , Cobre , Humanos , Metalotioneína , Saccharomyces cerevisiae/genética , TransactivadoresRESUMEN
This work describes the synthesis, enzymatic activities on PI3K and mTOR, in silico docking and cellular activities of various uncommon 2,4,7 trisubstituted pyrido[3,2-d]pyrimidines. The series synthesized offers a chemical diversity in C-7 whereas C-2 (3-hydroxyphenyl) and C-4 groups (morpholine) remain unchanged, in order to provide a better understanding of the molecular determinants of PI3K selectivity or dual activity on PI3K and mTOR. Some C-7 substituents were shown to improve the efficiency on kinases compared to the 2,4-di-substituted pyrimidopyrimidine derivatives used as references. Six novel derivatives possess IC50 values on PI3Kα between 3 and 10 nM. The compounds with the best efficiencies on PI3K and mTOR induced micromolar cytotoxicity on cancer cell lines possessing an overactivated PI3K pathway.
Asunto(s)
Diseño de Fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Neurofibromin is a large and multifunctional protein encoded by the tumor suppressor gene NF1, mutations of which cause the tumor predisposition syndrome neurofibromatosis type 1 (NF1). Over the last three decades, studies of neurofibromin structure, interacting partners, and functions have shown that it is involved in several cell signaling pathways, including the Ras/MAPK, Akt/mTOR, ROCK/LIMK/cofilin, and cAMP/PKA pathways, and regulates many fundamental cellular processes, such as proliferation and migration, cytoskeletal dynamics, neurite outgrowth, dendritic-spine density, and dopamine levels. The crystallographic structure has been resolved for two of its functional domains, GRD (GAP-related (GTPase-activating protein) domain) and SecPH, and its post-translational modifications studied, showing it to be localized to several cell compartments. These findings have been of particular interest in the identification of many therapeutic targets and in the proposal of various therapeutic strategies to treat the symptoms of NF1. In this review, we provide an overview of the literature on neurofibromin structure, function, interactions, and regulation and highlight the relationships between them.
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Regulación de la Expresión Génica , Neurofibromina 1/química , Neurofibromina 1/genética , Animales , Humanos , Neurofibromina 1/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death. In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by γ[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.
Asunto(s)
Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Anticuerpos/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Sistemas de Lectura Abierta , Fosforilación , Transducción de SeñalRESUMEN
LIMK2 is involved in neuronal functions by regulating actin dynamics. Different isoforms of LIMK2 are described in databanks. LIMK2a and LIMK2b are the most characterized. A few pieces of evidence suggest that LIMK2 isoforms might not have overlapping functions. In this study, we focused our attention on a less studied human LIMK2 isoform, LIMK2-1. Compared to the other LIMK2 isoforms, LIMK2-1 contains a supplementary C-terminal phosphatase 1 inhibitory domain (PP1i). We found out that this isoform was hominidae-specific and showed that it was expressed in human fetal brain and faintly in adult brain. Its coding sequence was sequenced in 173 patients with sporadic non-syndromic intellectual disability (ID), and we observed an association of a rare missense variant in the PP1i domain (rs151191437, p.S668P) with ID. Our results also suggest an implication of LIMK2-1 in neurite outgrowth and neurons arborization which appears to be affected by the p.S668P variation. Therefore our results suggest that LIMK2-1 plays a role in the developing brain, and that a rare variation of this isoform is a susceptibility factor in ID.
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Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Discapacidad Intelectual/metabolismo , Quinasas Lim/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Sistema Nervioso Central/citología , Predisposición Genética a la Enfermedad , Hominidae , Discapacidad Intelectual/genética , Quinasas Lim/genética , Ratones , Modelos Moleculares , Mutación Missense , Proyección Neuronal/fisiología , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas , Ratas , Homología de SecuenciaRESUMEN
LIMK1 and LIMK2 (LIMKs, LIM kinases) are kinases that play a crucial role in cytoskeleton dynamics by independently regulating both actin filament and microtubule remodeling. LIMK1 and, more recently, LIMK2 have been shown to be involved in cancer development and metastasis, resistance of cancer cells to microtubule-targeted treatments, neurological diseases, and viral infection. LIMKs have thus recently emerged as new therapeutic targets. Databanks describe three isoforms of human LIMK2: LIMK2a, LIMK2b, and LIMK2-1. Evidence suggests that they may not have completely overlapping functions. We biochemically characterized the three isoforms to better delineate their potential roles, focusing on LIMK2-1, which has only been described at the mRNA level in a single study. LIMK2-1 has a protein phosphatase 1 (PP1) inhibitory domain at its C-terminus which its two counterparts do not. We showed that the LIMK2-1 protein is indeed synthesized. LIMK2-1 does not phosphorylate cofilin, the canonical substrate of LIMKs, although it has kinase activity and promotes actin stress fiber formation. Instead, it interacts with PP1 and partially inhibits its activity towards cofilin. Our data suggest that LIMK2-1 regulates actin cytoskeleton dynamics by preventing PP1-mediated cofilin dephosphorylation, rather than by directly phosphorylating cofilin as its two counterparts, LIMK2a and LIMK2b. This specificity may allow for tight regulation of the phospho-cofilin pool, determining the fate of the cell.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Núcleo Celular/enzimología , Citoplasma/enzimología , Quinasas Lim/metabolismo , Transducción de Señal , Factores Despolimerizantes de la Actina/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Quinasas Lim/genética , Fosforilación , Proteína Fosfatasa 1/metabolismo , Interferencia de ARN , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator. CONCLUSIONS/SIGNIFICANCE: Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.
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Quinasas Lim/antagonistas & inhibidores , Neurofibromina 1/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Activación Enzimática , Células HEK293 , Células HeLa , Humanos , Quinasas Lim/metabolismo , Modelos Biológicos , Neurofibromina 1/química , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Fibras de Estrés/metabolismo , Técnicas del Sistema de Dos Híbridos , Quinasas Asociadas a rho/metabolismoRESUMEN
The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. To further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.
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Proteínas Portadoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/aislamiento & purificación , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Redes y Vías Metabólicas , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteómica , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Transducción de SeñalRESUMEN
Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.
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Quinasas Lim/metabolismo , Neuritas/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Cerebelo/enzimología , Hipocampo/enzimología , Isoenzimas/genética , Isoenzimas/metabolismo , Quinasas Lim/genética , Ratones , Neuritas/enzimología , Estructura Terciaria de Proteína , RatasRESUMEN
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line: CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using "in situ" proximity ligation assay (PLA).
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Astrocitos/enzimología , Núcleo Celular/enzimología , Neurofibromina 1/metabolismo , Astrocitoma/enzimología , Línea Celular Tumoral , Humanos , Complejos Multiproteicos/metabolismoRESUMEN
The Saccharomyces cerevisiae protein Tfs1p is known as a dual protein. On the one hand, it inhibits the carboxypeptidase Y protease, and on the other, it inhibits Ira2p, a GTPase-activating protein of Ras. We managed to dissect precise areas of Tfs1p specifically involved in only one of those functions. Based on these data, specific Tfs1p point mutants affected in only one of these two functions were constructed. In order to obtain insights on the physiological role of these functions, systematic phenotypic tests were performed on strains expressing these specific Tfs1p mutants. The results obtained demonstrate that the inhibition of Ira2p by Tfs1p is the predominant function under the conditions tested.
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Carboxipeptidasas/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Sustitución de Aminoácidos , Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Mutación Missense , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Tfs1p and Ylr179cp are yeast proteins belonging to the PEBP family. Tfs1p, but not Ylr179cp, has been shown to interact with and inhibit Ira2p, a GTPase-activating protein of Ras. Tfs1p has been shown to be a specific inhibitor of the CPY protease and the 3D structure of the complex has been resolved. To shed light on the molecular determinants of Tfs1p involved in the Tfs1/Ira2 interaction, the 3D structure of Ylr179cp has been modelled and compared to that of Tfs1p. Tfs1p point mutants and Tfs1 hybrid proteins combining regions of Tfs1p and Ylr179cp were also designed and their function was tested. Results, interpreted from a structural point of view, show that the accessibility of the surface pocket of Tfs1p, its N-terminal region and the specific electrostatic properties of a large surface region containing these two elements, play a crucial role in this interaction.
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Proteínas Activadoras de GTPasa/química , Modelos Moleculares , Ingeniería de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Secuencia de Bases , Cartilla de ADN , Proteínas Activadoras de GTPasa/metabolismo , Inmunoprecipitación , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl-CoA-dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity. They copurified with ceramide synthase activity, giving unequivocal evidence that they are subunits of the enzyme. In purified form, the acyl-CoA dependence, fatty acyl-CoA chain length specificity, and Fumonisin B1/Australifungin sensitivity of the ceramide synthase were the same as in cells, showing that these are properties of the enzyme and do not depend upon the membrane environment or other factors. SDS-PAGE analysis of purified ceramide synthase revealed the presence of a novel subunit of the enzyme, Lip1p. Lip1p is a single-span ER membrane protein that is required for ceramide synthesis in vivo and in vitro. The Lip1p regions required for ceramide synthesis are localized within the ER membrane or lumen.
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Proteínas de la Membrana/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Mutación/genética , Oxidorreductasas/aislamiento & purificación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Alineación de Secuencia , Esfingolípidos/biosíntesisRESUMEN
Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast, Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP.