RESUMEN
Reversible S-palmitoylation of cysteine residues critically controls transient membrane tethering of peripheral membrane proteins. Little is known about how the palmitoylation machinery governs their defined localization and function. We monitored the spatially resolved reaction dynamics and substrate specificity of the core mammalian palmitoylation machinery using semisynthetic substrates. Palmitoylation is detectable only on the Golgi, whereas depalmitoylation occurs everywhere in the cell. The reactions are not stereoselective and lack any primary consensus sequence, demonstrating that substrate specificity is not essential for de-/repalmitoylation. Both palmitate attachment and removal require seconds to accomplish. This reaction topography and rapid kinetics allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus. Unidirectional secretion ensures the maintenance of a proper steady-state protein distribution between the Golgi and the plasma membrane, which are continuous with endosomes. This generic spatially organizing system differs from conventional receptor-mediated targeting mechanisms and efficiently counteracts entropy-driven redistribution of palmitoylated peripheral membrane proteins over all membranes.
Asunto(s)
Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Lipoilación , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
BACKGROUND: Animal models of human inflammatory diseases have limited predictive quality for human clinical trials for various reasons including species specific activation mechanisms and the immunological background of the animals which markedly differs from the genetically heterogeneous and often aged patient population. OBJECTIVE: Development of an animal model allowing for testing therapeutics targeting pathways involved in the development of Atopic Dermatitis (AD) with better translatability to the patient. METHODS: NOD-scid IL2R γnull mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from AD and healthy volunteers were treated with IL-4 and the antagonistic IL-4 variant R121/Y124D (Pitrakinra). Levels of human (h)IgE, amount of B-, T- and plasma- cells and ratio of CD4 : CD8 positive cells served as read out for induction and inhibition of cell proliferation and hIgE secretion. Results were compared to in vitro analysis. RESULTS: hIgE secretion was induced by IL-4 and inhibited by the IL-4 antagonist Pitrakinra in vivo when formulated with methylcellulose. B-cells proliferated in response to IL-4 in vivo; the effect was abrogated by Pitrakinra. IL-4 shifted CD4 : CD8 ratios in vitro and in vivo when hPBMC derived from healthy volunteers were used. Pitrakinra reversed the effect. Human PBMC derived from patients with AD remained inert and engrafted mice reflected the individual responses observed in vitro. CONCLUSION: NOD-scid IL2R γnull mice engrafted with human PBMC reflect the immunological history of the donors and provide a complementary tool to in vitro studies. Thus, studies in this model might provide data with better translatability from bench to bedside.
Asunto(s)
Subunidad gamma Común de Receptores de Interleucina/genética , Monocitos/metabolismo , Transducción de Señal , Animales , Citometría de Flujo , Humanos , Inmunoglobulina E/metabolismo , Interleucina-4/metabolismo , Metilcelulosa/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCIDRESUMEN
The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.
Asunto(s)
Actinas/química , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Cristalización , Cristalografía por Rayos X/métodos , Forminas , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Propiedades de SuperficieRESUMEN
The actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2.
Asunto(s)
Proteínas Fetales/química , Proteínas de Microfilamentos/química , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Secuencia de Aminoácidos , Animales , Anisotropía , Proliferación Celular , Forminas , Células HeLa , Humanos , Microscopía Fluorescente/métodos , Modelos Biológicos , Datos de Secuencia Molecular , Oogénesis , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Prenilación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Dicroismo Circular , Farnesiltransferasa/metabolismo , Mutación/genética , Peroxinas , Unión Proteica , Señales de Clasificación de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Relación Estructura-ActividadRESUMEN
Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.
Asunto(s)
Luz , Lipoproteínas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Terpenos/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biología Computacional , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Lipoproteínas/síntesis química , Lipoproteínas/química , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas ras/síntesis química , Proteínas ras/químicaRESUMEN
During the last years, post-translational modification of peripheral membrane proteins with hydrophobic side groups has been attributed to a couple of additional functions than just simple anchoring into lipid bilayers. In particular isoprenylation and N- and S-acylation did quicken interest in terms of specific recognition elements for protein-protein interactions and as hydrophobic switches that allow for temporal regulated association with distinct target structures. Furthermore new insights into the heterogeneity of natural membranes have connected the physical properties of e.g. farnesyl or palmitoyl side chains with a preference for such sub-compartments as lipid rafts or caveolae. In this review the impact of the two frequently realized modifications by isoprenylation and S-acylation on the process of cellular signal transduction is exemplified with proteins of the Ras and Rab family of small GTP-binding proteins.
Asunto(s)
Ácidos Grasos/metabolismo , Microdominios de Membrana/fisiología , Prenilación de Proteína , Procesamiento Proteico-Postraduccional , Terpenos/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transferasas Alquil y Aril/metabolismo , Animales , Inhibidores de Disociación de Guanina Nucleótido/fisiología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inteínas/fisiología , Lipoproteínas/síntesis química , Modelos Moleculares , Mapeo de Interacción de Proteínas , Transducción de Señal , Transferasas , Proteínas ras/química , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
The oncoprotein Ras is anchored in lipid membranes due to its C-terminal lipid modification. The ubiquitously expressed Ras nucleotide exchange-factor hSOS1 promotes nucleotide exchange and thus Ras activation. This reaction is enhanced by a positive feedback loop whereby activated Ras binds to an allosteric site of SOS to enhance GEF activity. Here we present biochemical data showing that prenylation of both active site bound and allosterically bound N-Ras is required for efficient hSOS1-promoted nucleotide exchange. Our results indicate that prenyl sensitivity of the allosteric feedback-activation is mediated by the PH domain of hSOS1. Farnesylation of Ras thereby allows hSOS1 to bind even GDP-loaded allosteric regulator to maintain basal hSOS1-activity.