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Isabel Merida - One of the best experts on this subject based on the ideXlab platform.
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interplay between SNX27 and dag metabolism in the control of trafficking and signaling at the is
International Journal of Molecular Sciences, 2020Co-Authors: Natalia Gonzalezmancha, Isabel MeridaAbstract:Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell–cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell–cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell–cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell–APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.
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diacylglycerol kinase control of protein kinase c
Biochemical Journal, 2019Co-Authors: Isabel Merida, Cristina Rodriguezrodriguez, Javier Arranznicolas, Antonia AvilafloresAbstract:The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell-cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.
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sorting nexin 27 interactome in t lymphocytes identifies zona occludens 2 dynamic redistribution at the immune synapse
Traffic, 2017Co-Authors: Maria Tellolafoz, Severine I Gharbi, Gonzalo Martinezmartinez, Cristina Rodriguezrodriguez, Juan Pablo Albar, Morgan Huse, Isabel MeridaAbstract:T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO-1 (PDZ) domain-interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T-lymphocytes, SNX27-positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS-forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild-type and a mutant-deficient for PDZ cargo recognition identified the epithelial cell-cell junction protein zona occludens-2 (ZO-2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO-2 PDZ-dependent interaction, and demonstrated its role controlling the dynamic localization of ZO-2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.
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a molecular code for endosomal recycling of phosphorylated cargos by the SNX27 retromer complex
Nature Structural & Molecular Biology, 2016Co-Authors: Thomas Clairfeuille, Zhe Yang, Markus C Kerr, Mintu Chandra, Audrey S M Chan, Maria Tellolafoz, Caroline Mas, Jocelyn Widagdo, Blessy Paul, Isabel MeridaAbstract: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 β2 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.
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phosphoinositide binding by the SNX27 ferm domain regulates its localization at the immune synapse of activated t cells
Journal of Cell Science, 2014Co-Authors: Rajesh Ghai, Rohan D Teasdale, Thomas Clairfeuille, Suzanne J Norwood, Isabel Merida, Zhe Yang, Maria Tellolafoz, Brett M CollinsAbstract: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.
Paul A Slesinger - One of the best experts on this subject based on the ideXlab platform.
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girk currents in vta dopamine neurons control the sensitivity of mice to cocaine induced locomotor sensitization
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Robert Rifkin, Deborah Huyghe, Manasa Parakala, Erin Aisenberg, Stephen J Moss, Paul A SlesingerAbstract:GABABR-dependent activation of G protein-gated inwardly rectifying potassium channels (GIRK or KIR3) provides a well-known source of inhibition in the brain, but the details on how this important inhibitory pathway affects neural circuits are lacking. We used sorting nexin 27 (SNX27), an endosomal adaptor protein that associates with GIRK2c and GIRK3 subunits, to probe the role of GIRK channels in reward circuits. A conditional knockout of SNX27 in both substantia nigra pars compacta and ventral tegmental area (VTA) dopamine neurons leads to markedly smaller GABABR- and dopamine D2R-activated GIRK currents, as well as to suprasensitivity to cocaine-induced locomotor sensitization. Expression of the SNX27-insensitive GIRK2a subunit in SNX27-deficient VTA dopamine neurons restored GIRK currents and GABABR-dependent inhibition of spike firing, while also resetting the mouse’s sensitivity to cocaine-dependent sensitization. These results establish a link between slow inhibition mediated by GIRK channels in VTA dopamine neurons and cocaine addiction, revealing a therapeutic target for treating addiction.
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sorting nexin 27 regulation of g protein gated inwardly rectifying k channels attenuates in vivo cocaine response
Neuron, 2014Co-Authors: Paul A Slesinger, Michaelanne B MunozAbstract:The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.
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ras association domain of sorting nexin 27 is critical for regulating expression of girk potassium channels
PLOS ONE, 2013Co-Authors: Bartosz Balana, Laia Bahima, Karthik Bodhinathan, Jaume Taura, Natalie Taylor, Margaret Nettleton, Francisco Ciruela, Paul A SlesingerAbstract:G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.
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ras associated ra domain of sorting nexin 27 SNX27 is critical for regulating girk channels
Biophysical Journal, 2012Co-Authors: Bartosz Balana, Laia Bahima, Karthik Bodhinathan, Margaret Nettleton, Francisco Ciruela, Natalie M Taylor, Paul A SlesingerAbstract:G-protein-gated potassium inwardly rectifying (GIRK) channels play an important role in regulating neuronal excitability in the brain. We have previously shown that trafficking and surface expression of GIRK channels are regulated by sorting nexin 27 (SNX27). SXN27 contains three domains; a phosphoinositide-binding Phox (PX) domain that targets SNX27 preferentially to early endosomes, a PDZ domain that associates directly with carboxyl-terminal tailof GIRK3 and GIRK2c subunits, and a Ras-associated RA domain that could potentially bind to small monomeric G proteins. The functional consequence of this interaction with the RA domain was unknown. Here we show for the first time that expression of a SNX27 mutant devoid of the RA domain (SNX27ΔRA) in HEK293T cells abrogates the down-regulation of baclofen-induced GIRK2c/3 currents that is typically observed with wild-type SNX27 (12.4±3.6 pA/pF (SNX27 WT group) vs. 55.8±8.2 pA/pF (SNX27ΔRA) vs. 47.1±6.7pA/pF (control group without SNX27); mean current density±SEM). Using bimolecular fluorescence complementation (BiFC) technique to follow the intracellular localization of GIRK2c/3 heterotetramers, we observed that SNX27 but not SNX27ΔRA increases the number of GIRK2c/3 heterotetramers in early endosomes. Moreover, a single point mutation within the RA domain (K305A) was sufficient to disrupt SNX27-dependent clustering of GIRK2c/3 heterotetramers, mimicking the functional effects of the RA deletion. Recently, it was shown that the RA domain of SNX27 interacts with H-Ras in vitro. We postulate that K305A disrupts RA interaction with H-Ras. In conclusion, we provide the first functional evidence that intact SNX27 RA domain and interaction with small monomeric G proteins is necessary for the regulation of GIRK channels. These new findings suggest that H-Ras signaling directly affects the magnitude of GIRK currents through regulation of SNX27.
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mechanism underlying selective regulation of g protein gated inwardly rectifying potassium channels by the psychostimulant sensitive sorting nexin 27
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Bartosz Balana, Laia Bahima, Innokentiy Maslennikov, Witek Kwiatkowski, Kalyn M Stern, Senyon Choe, Paul A SlesingerAbstract:G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.
Brett M Collins - One of the best experts on this subject based on the ideXlab platform.
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biochemical basis for an interaction between SNX27 and the flexible snx1 n terminus
Advances in biological regulation, 2021Co-Authors: Mintu Chandra, Brett M Collins, Lauren P JacksonAbstract:Abstract Metazoans require the sorting nexin (SNX) protein, SNX27, to recycle hundreds of important transmembrane protein receptors from endosomes to the plasma membrane. Cargo recycling by SNX27 requires its interaction with retromer, a heterotrimer known to assemble on membranes with multiple sorting nexins, including SNX-BAR proteins and SNX3. SNX27 has also been functionally linked to SNX-BARs, but the molecular basis of this interaction has been unknown. We identify a direct biochemical interaction between the conserved and flexible SNX1/SNX2 N-terminus and full-length SNX27 using purified proteins in pulldown experiments. Sequence alignments indicate both SNX1 and SNX2 contain two short and conserved stretches of acidic residues bearing a DxF motif in their flexible N-terminal regions. Biochemical pulldown and mapping experiments reveal forty residues in the N-terminus of either SNX1 or SNX2 can mediate binding to SNX27. SNX27 truncation analysis demonstrates the SNX27 FERM domain binds the SNX1 N-terminus. Calorimetry experiments quantified binding between the SNX1 N-terminus and SNX27 in the low micromolar affinity range (KD ∼10 μM) and suggest the second DxF motif may play a more prominent role in binding. Mutation of either DxF sequence in SNX1 abrogates measurable binding to SNX27 in the calorimeter. Modelling from both predicted and experimentally determined structures suggests the SNX27 FERM domain could accommodate both DxF motifs simultaneously. Together, these data suggest SNX27 is directly linked to specific SNX-BAR proteins through binding acidic motifs in the SNX1 or SNX2 N-terminus.
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mechanistic basis for SNX27 retromer coupling to escpe 1 in promoting endosomal cargo recycling
bioRxiv, 2021Co-Authors: Boris Simonetti, Brett M Collins, Chris M Danson, Ashley J Evans, Qian Guo, Manuel Gimenezandres, Kaien Chen, Edmund R R Moody, T Williams, Peter J CullenAbstract:ABSTRACT Sorting nexin-27 (SNX27)-Retromer is an endosomal sorting complex that orchestrates endosome-to-plasma membrane recycling of hundreds of internalized receptors, channels and transporters, enzymes and adhesion molecules. While SNX27-Retromer is essential for development, subtle functional defects are observed in human disease, most notably neurodegenerative and neurological disorders. Achieving a thorough mechanistic dissection of SNX27-Retromer is central to understanding endosomal sorting in health and disease. Here we combine biochemical, structural and cellular analyses to establish the mechanistic basis through which SNX27-Retromer couples to the membrane tubulating ESCPE-1 complex (Endosomal SNX-BAR sorting complex for promoting exit 1). We show that a conserved surface in the FERM (4.1/ezrin/radixin/moesin) domain of SNX27 directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the disordered amino-termini of the SNX1 and SNX2 subunits of ESCPE-1. This interaction hands-over SNX27-Retromer captured integral membrane proteins into ESCPE-1 tubular profiles to promote their cell surface recycling. Through phylogenetic analysis, we reveal that SNX27:Retromer:ESCPE-1 assembly evolved in a stepwise manner during the early evolution of metazoans, which reflects the increasing complexity of endosomal sorting from the ancestral opisthokont to modern animals.
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downregulation of SNX27 expression does not exacerbate amyloidogenesis in the app ps1 alzheimer s disease mouse model
Neurobiology of Aging, 2019Co-Authors: Michael R Milne, Rohan D Teasdale, Brett M Collins, Genevieve Kinna, Lei Qian, Marion T Turnbull, Elizabeth J CoulsonAbstract:Abstract There is in vitro evidence that sorting nexin family member 27 (SNX27), a member of the retromer complex, changes the distribution of the amyloid-beta (Aβ) precursor protein (APP) to promote its recycling and thereby prevent the production of Aβ, the toxic protein associated with Alzheimer's disease (AD). In this study, we analyzed the phenotype of the familial AD APP/PS mouse strain lacking one copy of the SNX27 gene. The reduction in SNX27 expression had no significant effect on the in vivo accumulation of soluble, total, or plaque-deposited Aβ, which is overproduced by the familial APP/PS transgenes. Hippocampal structure and cholinergic basal forebrain neuronal health were also unaffected. Nonetheless, mild positive and negative effects of age and/or genotype on spatial navigation performance were observed in SNX27+/− and SNX27+/−APP/PS mice, respectively. These data suggest that downregulation of SNX27 alone does not have long-term negative consequences on spatial memory, but that cognitive dysfunction in the context of high Aβ deposition is exacerbated by the cellular or molecular changes induced by reduced SNX27 function.
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sorting nexin 27 couples pthr trafficking to retromer for signal regulation in osteoblasts during bone growth
Molecular Biology of the Cell, 2016Co-Authors: Audrey S M Chan, Rohan D Teasdale, Thomas Clairfeuille, Wanjin Hong, Euphemie Landaobassonga, Genevieve Kinna, Li Shen Loo, Tak Sum Cheng, Minghao Zheng, Brett M CollinsAbstract: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.
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structure and membrane binding properties of the endosomal tetratricopeptide repeat tpr domain containing sorting nexins snx20 and snx21
Journal of Biological Chemistry, 2015Co-Authors: Thomas Clairfeuille, Rohan D Teasdale, Suzanne J Norwood, Brett M CollinsAbstract: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.
Wanjin Hong - One of the best experts on this subject based on the ideXlab platform.
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sorting nexin 27 SNX27 a novel regulator of cystic fibrosis transmembrane conductance regulator cftr trafficking
bioRxiv, 2018Co-Authors: M I Mcdermott, Wanjin Hong, William R Thelin, Yunjia Chen, P T Lyons, G Reilly, Martina Gentzsch, C Lei, Stutts M Jackson, M P PlayfordAbstract:The underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.
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sorting nexin 27 couples pthr trafficking to retromer for signal regulation in osteoblasts during bone growth
Molecular Biology of the Cell, 2016Co-Authors: Audrey S M Chan, Rohan D Teasdale, Thomas Clairfeuille, Wanjin Hong, Euphemie Landaobassonga, Genevieve Kinna, Li Shen Loo, Tak Sum Cheng, Minghao Zheng, Brett M CollinsAbstract: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.
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sorting nexin 27 regulates aβ production through modulating γ secretase activity
Cell Reports, 2014Co-Authors: Xin Wang, Timothy Y. Huang, Yingjun Zhao, Qiuyang Zheng, Robert Thompson, Yunwu Zhang, Wanjin HongAbstract:Summary Patients with Down syndrome (DS) invariably develop Alzheimer's disease (AD) pathology in their 40s. We have recently found that overexpression of a chromosome 21-encoded microRNA-155 results in decreased levels of the membrane trafficking component, SNX27, diminishing glutamate receptor recycling and thereby impairing synaptic functions in DS. Here, we report a function of SNX27 in regulating β-amyloid (Aβ) generation by modulating γ-secretase activity. Downregulation of SNX27 using RNAi increased Aβ production, whereas overexpression of full-length SNX27, but not SNX27ΔPDZ, reversed the RNAi-mediated Aβ elevation. Moreover, genetic deletion of SNX27 promoted Aβ production and neuronal loss, whereas overexpression of SNX27 using an adeno-associated viral (AAV) vector reduced hippocampal Aβ levels in a transgenic AD mouse model. SNX27 associates with the γ-secretase complex subunit presenilin 1; this interaction dissociates the γ-secretase complex, thus decreasing its proteolytic activity. Our study establishes a molecular mechanism for Aβ-dependent pathogenesis in both DS and AD.
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a role for sorting nexin 27 in ampa receptor trafficking
Nature Communications, 2014Co-Authors: Ning Tang, Muthafar Alhaddawi, Gavin S Dawe, Wanjin HongAbstract:Sorting nexin 27 (SNX27) regulates endosomal sorting of glutamate receptors. Loo et al. show that SNX27 is localized to recycling endosomes within dendritic spines where it interacts with glutamate receptors, allowing them to be shuttled to the postsynaptic membrane.
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deficiency of sorting nexin 27 SNX27 leads to growth retardation and elevated levels of n methyl d aspartate receptor 2c nr2c
Molecular and Cellular Biology, 2011Co-Authors: Lei Cai, Li Shen Loo, Vadim Atlashkin, Brendon J Hanson, Wanjin HongAbstract:Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27−/− neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.
Peter J Cullen - One of the best experts on this subject based on the ideXlab platform.
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mechanistic basis for SNX27 retromer coupling to escpe 1 in promoting endosomal cargo recycling
bioRxiv, 2021Co-Authors: Boris Simonetti, Brett M Collins, Chris M Danson, Ashley J Evans, Qian Guo, Manuel Gimenezandres, Kaien Chen, Edmund R R Moody, T Williams, Peter J CullenAbstract:ABSTRACT Sorting nexin-27 (SNX27)-Retromer is an endosomal sorting complex that orchestrates endosome-to-plasma membrane recycling of hundreds of internalized receptors, channels and transporters, enzymes and adhesion molecules. While SNX27-Retromer is essential for development, subtle functional defects are observed in human disease, most notably neurodegenerative and neurological disorders. Achieving a thorough mechanistic dissection of SNX27-Retromer is central to understanding endosomal sorting in health and disease. Here we combine biochemical, structural and cellular analyses to establish the mechanistic basis through which SNX27-Retromer couples to the membrane tubulating ESCPE-1 complex (Endosomal SNX-BAR sorting complex for promoting exit 1). We show that a conserved surface in the FERM (4.1/ezrin/radixin/moesin) domain of SNX27 directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the disordered amino-termini of the SNX1 and SNX2 subunits of ESCPE-1. This interaction hands-over SNX27-Retromer captured integral membrane proteins into ESCPE-1 tubular profiles to promote their cell surface recycling. Through phylogenetic analysis, we reveal that SNX27:Retromer:ESCPE-1 assembly evolved in a stepwise manner during the early evolution of metazoans, which reflects the increasing complexity of endosomal sorting from the ancestral opisthokont to modern animals.
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retromer controls planar polarity protein levels and asymmetric localization at intercellular junctions
Current Biology, 2019Co-Authors: Helen Strutt, Neil David Pearson, Kirsty J Mcmillan, Paul F Langton, David Strutt, Peter J CullenAbstract:The coordinated polarization of cells in the plane of a tissue, termed planar polarity, is a characteristic feature of epithelial tissues [1]. In the fly wing, trichome positioning is dependent on the core planar polarity proteins adopting asymmetric subcellular localizations at apical junctions, where they form intercellular complexes that link neighboring cells [1-3]. Specifically, the seven-pass transmembrane protein Frizzled and the cytoplasmic proteins Dishevelled and Diego localize to distal cell ends, the four-pass transmembrane protein Strabismus and the cytoplasmic protein Prickle localize proximally, and the seven-pass transmembrane spanning atypical cadherin Flamingo localizes both proximally and distally. To establish asymmetry, these core proteins are sorted from an initially uniform distribution; however, the mechanisms underlying this polarized trafficking remain poorly understood. Here, we describe the identification of retromer, a master controller of endosomal recycling [4-6], as a key component regulating core planar polarity protein localization in Drosophila. Through generation of mutants, we verify that loss of the retromer-associated SNX27 cargo adaptor, but notably not components of the Wash complex, reduces junctional levels of the core proteins Flamingo and Strabismus in the developing wing. We establish that SNX27 directly associates with Flamingo via its C-terminal PDZ binding motif, and we show that SNX27 is essential for normal Flamingo trafficking. We conclude that Wash-independent retromer function and the SNX27 cargo adaptor are important components in the endosomal recycling of Flamingo and Strabismus back to the plasma membrane and thus contribute to the establishment and maintenance of planar polarization.
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sorting nexin 21 is a scaffold for the endosomal recruitment of huntingtin
Journal of Cell Science, 2018Co-Authors: Chris M Danson, Neil David Pearson, Kate J Heesom, Peter J CullenAbstract:ABSTRACT The endo-lysosomal network serves an essential role in determining the fate of endocytosed transmembrane proteins and their associated proteins and lipids. Sorting nexins (SNXs) play a central role in the functional organisation of this network. Comprising over 30 proteins in humans, SNXs are classified into sub-groups based on the presence of additional functional domains. Sorting nexin-20 (SNX20) and sorting nexin-21 (SNX21) comprise the SNX-PXB proteins. The presence of a predicted protein-protein interaction domain, termed the PX-associated B (PXB) domain, has led to the proposal that they function as endosome-associated scaffolds. Here, we used unbiased quantitative proteomics to define the SNX21 interactome. We reveal that the N-terminal extension of SNX21 interacts with huntingtin (Htt) whereas the PXB domain appears to associate with septins, a family of cytoskeletal- and membrane-associated proteins. In establishing that these interactions are sufficient for SNX21 to recruit Htt and septins on to an endosomal population, we reveal a scaffolding function for this sorting nexin. Our work paves the way for a more-detailed mechanistic analysis of the role(s) of the SNX-PXB proteins in endosomal biology.
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retromer and wash dependent sorting of nutrient transporters requires a multivalent interaction network with ankrd50
Journal of Cell Science, 2016Co-Authors: Arunas Kvainickas, Peter J Cullen, Kate J Heesom, Ana Jimenez Orgaz, Heike Nagele, Britta Diedrich, Jorn Dengjel, Florian SteinbergAbstract:ABSTRACT Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson9s-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer9s association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27–retromer–WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27–retromer–WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins.
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a unique pdz domain and arrestin like fold interaction reveals mechanistic details of endocytic recycling by SNX27 retromer
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Matthew Gallon, Richard B Sessions, Rajesh Ghai, Rohan D Teasdale, Brett M Collins, Thomas Clairfeuille, Florian Steinberg, Peter J CullenAbstract: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 β-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.