RESUMEN
Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
Asunto(s)
Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Lipopolisacáridos , Proteínas de Transporte de Membrana , Acinetobacter/química , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana , Conformación Proteica/efectos de los fármacos , Especificidad por SustratoRESUMEN
Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.
Asunto(s)
Antibacterianos , Lipopolisacáridos , Proteínas de Transporte de Membrana , Animales , Humanos , Ratones , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/clasificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Desarrollo de MedicamentosRESUMEN
Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4-10. NALCN activity is under tight regulation11-14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.
Asunto(s)
Microscopía por Crioelectrón , Canales Iónicos/química , Canales Iónicos/ultraestructura , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Mutación con Ganancia de Función , Células HEK293 , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación Missense , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.
Asunto(s)
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidad , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencias de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Estabilidad de Enzimas , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Genes Esenciales , Hidrolasas/química , Hidrolasas/metabolismo , Lípido A/química , Lípido A/metabolismo , Lipopolisacáridos/biosíntesis , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Periplasma/química , Periplasma/metabolismo , Unión Proteica , VirulenciaRESUMEN
Electron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. However, the sample preparation is still the bottleneck in the cryogenic electron microscopy (cryo-EM) workflow. Classical specimen preparation methods employ a harsh paper-blotting step, and the protein particles are exposed to a damaging air-water interface. Therefore, improved preparation strategies are urgently needed. Here, we present an amended microfluidic sample preparation method, which entirely avoids paper blotting and allows the passivation of the air-water interface during the preparation process. First, a climate jet excludes oxygen from the sample environment and controls the preparation temperature by varying the relative humidity of the grid environment. Second, the integrated "coverslip injector" allows the modulation of the air-water interface of the thin sample layer with effector molecules. We will briefly discuss the climate jet's effect on the stability and dynamics of the sample thin films. Furthermore, we will address the coverslip injector and demonstrate significant improvement in the sample quality.
Asunto(s)
Proteínas , Manejo de Especímenes , Microscopía por Crioelectrón , Microfluídica , AguaRESUMEN
Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of sorting nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid-stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Glutamina/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Nexinas de Clasificación/fisiología , Autofagia , Ciclo Celular , Proliferación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Dominios PDZ , Transporte de Proteínas/fisiología , Transducción de Señal , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Fracciones Subcelulares/metabolismoRESUMEN
Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsP-binding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi- and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.
Asunto(s)
Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos/inmunología , Fosfatos de Fosfatidilinositol/metabolismo , Nexinas de Clasificación/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Membrana Celular/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Células Jurkat , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Transducción de SeñalRESUMEN
The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed ß-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.
Asunto(s)
Endocitosis/fisiología , Dominios PDZ/genética , Nexinas de Clasificación/química , Proteínas de Transporte Vesicular/química , Secuencia de Aminoácidos , Animales , Arrestina/química , Arrestina/genética , Encefalopatías/genética , Encefalopatías/metabolismo , Encefalopatías/patología , Cristalografía por Rayos X , Endosomas/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Pliegue de Proteína , Señales de Clasificación de Proteína/genética , ARN Interferente Pequeño/genética , Ratas , Homología de Secuencia de Aminoácido , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds.
Asunto(s)
Endosomas/metabolismo , Nexinas de Clasificación/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Endosomas/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilinositoles/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Nexinas de Clasificación/análisis , Nexinas de Clasificación/metabolismo , Difracción de Rayos XRESUMEN
The endosome-associated cargo adaptor sorting nexin-27 (SNX27) is linked to various neuropathologies through sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies, we performed proteomics in rat primary neurons to identify SNX27-dependent cargoes, and identified proteins linked to excitotoxicity, epilepsy, intellectual disabilities, and working memory deficits. Focusing on the synaptic adhesion molecule LRFN2, we established that SNX27 binds to LRFN2 and regulates its endosomal sorting. Furthermore, LRFN2 associates with AMPA receptors and knockdown of LRFN2 results in decreased surface AMPA receptor expression, reduced synaptic activity, and attenuated hippocampal long-term potentiation. Overall, our study provides an additional mechanism by which SNX27 can control AMPA receptor-mediated synaptic transmission and plasticity indirectly through the sorting of LRFN2 and offers molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Endosomas/metabolismo , Hipocampo/metabolismo , Humanos , Potenciación a Largo Plazo , Trastornos de la Memoria/metabolismo , Transporte de Proteínas , Proteómica/métodos , Ratas , Transmisión SinápticaRESUMEN
Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Cromatografía en Gel , Proteínas Fluorescentes Verdes/genética , Histidina/química , Histidina/metabolismo , Humanos , Proteínas de la Membrana/genética , Oligopéptidos/química , Oligopéptidos/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Animales , Cucarachas , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Dominios Proteicos , Proteínas Recombinantes de Fusión/químicaRESUMEN
Voltage-gated sodium (Nav) channels initiate and propagate action potentials in excitable cells, and are frequently dysregulated or mutated in human disease. Despite decades of intense physiological and biophysical research, eukaryotic Nav channels have so far eluded high-resolution structure determination because of their biochemical complexity. Recently, simpler bacterial voltage-gated sodium (BacNav) channels have provided templates to understand the structural basis of voltage-dependent activation, inactivation, ion selectivity, and drug block in eukaryotic Nav and related voltage-gated calcium (Cav) channels. Further breakthroughs employing BacNav channels have also enabled visualization of bound small molecule modulators that can guide the rational design of next generation therapeutics. This review will highlight the emerging structural biology of BacNav channels and its contribution to our understanding of the gating, ion selectivity, and pharmacological regulation of eukaryotic Nav (and Cav) channels.
Asunto(s)
Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , PorosidadRESUMEN
The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development.
Asunto(s)
Desarrollo Óseo/fisiología , Osteoblastos/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Desarrollo Óseo/genética , Remodelación Ósea/fisiología , Calcificación Fisiológica/genética , Calcificación Fisiológica/fisiología , Endosomas/metabolismo , Células HEK293/metabolismo , Humanos , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Osteoblastos/efectos de los fármacos , Dominios PDZ , Hormona Paratiroidea/farmacología , Transporte de Proteínas , Receptor de Hormona Paratiroídea Tipo 1/genética , Transducción de Señal , Nexinas de Clasificación/genéticaRESUMEN
Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95-Dlg-ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the ß2 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor, lack these sequence determinants. Instead, we identified conserved sites of phosphorylation that substitute for acidic residues and dramatically enhance SNX27 interactions. This newly identified mechanism suggests a likely regulatory switch for PDZ interaction and protein transport by the SNX27-retromer complex. Defining this SNX27 binding code allowed us to classify more than 400 potential SNX27 ligands with broad functional implications in signal transduction, neuronal plasticity and metabolite transport.
Asunto(s)
Endosomas/metabolismo , Nexinas de Clasificación/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Dominios PDZ , Fosforilación , Unión Proteica , Mapas de Interacción de Proteínas , Transporte de Proteínas , Receptores de Glutamato/metabolismo , Alineación de Secuencia , Transducción de Señal , Nexinas de Clasificación/químicaRESUMEN
The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand-containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism.