163 results on '"Peter J. Cullen"'
Search Results
2. Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health
- Author
-
James L. Daly, Chris M. Danson, Philip A. Lewis, Lu Zhao, Sara Riccardo, Lucio Di Filippo, Davide Cacchiarelli, Daehoon Lee, Stephen J. Cross, Kate J. Heesom, Wen-Cheng Xiong, Andrea Ballabio, James R. Edgar, Peter J. Cullen, Daly, James L [0000-0002-4551-1256], Lewis, Philip A [0000-0002-2868-2459], Cacchiarelli, Davide [0000-0002-9621-6716], Cross, Stephen J [0000-0003-3565-0479], Heesom, Kate J [0000-0002-5418-5392], Xiong, Wen-Cheng [0000-0001-9071-7598], Ballabio, Andrea [0000-0003-1381-4604], Edgar, James R [0000-0001-7903-8199], Cullen, Peter J [0000-0002-9070-8349], and Apollo - University of Cambridge Repository
- Subjects
Proteomics ,Protein Transport ,Multidisciplinary ,Proteome ,General Physics and Astronomy ,Humans ,General Chemistry ,Endosomes ,Multiomics ,Lysosomes ,General Biochemistry, Genetics and Molecular Biology ,Neuroprotection - Abstract
Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.
- Published
- 2023
3. Ship Source Pollution Regimes (Canada)—A Primer
- Author
-
Peter J. Cullen
- Published
- 2023
4. Structure of the Commander Endosomal Trafficking Complex Linked to X-Linked Intellectual Disability/Ritscher-Schinzel Syndrome
- Author
-
Michael D. Healy, Kerrie E. McNally, Rebeka Butkovic, Molly Chilton, Kohji Kato, Joanna Sacharz, Calum McConville, Edmund R.R. Moody, Shrestha Shaw, Vicente J. Planelles-Herrero, Sathish K.N. Yadav, Jennifer Ross, Ufuk Borucu, Catherine S. Palmer, Kai-En Chen, Tristan I. Croll, Ryan J. Hall, Nikeisha J. Caruana, Rajesh Ghai, Thi H.D. Nguyen, Kate J. Heesom, Shinji Saitoh, Imre Berger, Christiane Schaffitzel, Tom A. Williams, David A. Stroud, Emmanuel Derivery, Brett M. Collins, and Peter J. Cullen
- Abstract
SUMMARYThe Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy andin silicopredictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.
- Published
- 2023
5. CHC22 clathrin membrane recruitment uses SNX5 in bipartite interaction with secretory tether p115
- Author
-
Joshua Greig, George T. Bates, Daowen I. Yin, Boris Simonetti, Peter J. Cullen, and Frances M. Brodsky
- Abstract
The two clathrin isoforms, CHC17 and CHC22, generate separate vesicles for intracellular transport. CHC17 mediates endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, transports the glucose transporter GLUT4 from the endoplasmic-reticulum-to-Golgi intermediate compartment (ERGIC) to an intracellular GLUT4 storage compartment (GSC) from where GLUT4 is mobilized by insulin. Molecular determinants distinguishing the trafficking of CHC22 clathrin from CHC17 within the GLUT4 pathway are defined in this study. The C-terminal trimerization domain of CHC22, but not CHC17, directly binds SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. Both the SNX5-BAR domain and an isoform-specific patch on the CHC22 N-terminal domain separately mediate binding to p115, and both interactions are required for CHC22 recruitment. These indirect and direct interactions at each CHC22 terminus are required for GLUT4 traffic to the GSC, defining a dual mechanism regulating the function of CHC22 in glucose metabolism.Summary statementCHC22 clathrin uses a bipartite mechanism for recruitment to the early secretory pathway, where it targets the GLUT4 transporter to an insulin-responsive intracellular compartment. Localization requires binding to the ERGIC tether p115 through sorting nexin 5 interaction at the CHC22 C-terminus and directly via the CHC22 N-terminal domain.
- Published
- 2022
6. Multiomic Approach Characterises the Neuroprotective Role of Retromer in Regulating Lysosomal Health
- Author
-
James L. Daly, Chris M. Danson, Philip A. Lewis, Sara Riccardo, Lucio Di Filippo, Davide Cacchiarelli, Stephen J. Cross, Kate J. Heesom, Andrea Ballabio, James R. Edgar, and Peter J. Cullen
- Abstract
Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling late-stage maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform the first integrated multiomics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify profound changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide an unprecedented integrated view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.
- Published
- 2022
7. Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome
- Author
-
Michael D. Healy, Kerrie E. McNally, Rebeka Butkovič, Molly Chilton, Kohji Kato, Joanna Sacharz, Calum McConville, Edmund R.R. Moody, Shrestha Shaw, Vicente J. Planelles-Herrero, Sathish K.N. Yadav, Jennifer Ross, Ufuk Borucu, Catherine S. Palmer, Kai-En Chen, Tristan I. Croll, Ryan J. Hall, Nikeisha J. Caruana, Rajesh Ghai, Thi H.D. Nguyen, Kate J. Heesom, Shinji Saitoh, Imre Berger, Christiane Schaffitzel, Tom A. Williams, David A. Stroud, Emmanuel Derivery, Brett M. Collins, and Peter J. Cullen
- Subjects
General Biochemistry, Genetics and Molecular Biology - Published
- 2023
8. ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1
- Author
-
Boris Simonetti, James L. Daly, Lorena Simón-Gracia, Katja Klein, Saroja Weeratunga, Carlos Antón-Plágaro, Allan Tobi, Lorna Hodgson, Phil Lewis, Kate J. Heesom, Deborah K. Shoemark, Andrew D. Davidson, Brett M. Collins, Tambet Teesalu, Yohei Yamauchi, and Peter J. Cullen
- Subjects
Proteomics ,Multidisciplinary ,SARS-CoV-2 ,viruses ,COVID-19 ,Endosomes ,sorting nexin ,Neuropilin-1 ,Host-Pathogen Interactions ,Spike Glycoprotein, Coronavirus ,Humans ,Nanoparticles ,CRISPR-Cas Systems ,Sorting Nexins ,endosome ,Gene Deletion - Abstract
Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed “cargoes”) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2., Proceedings of the National Academy of Sciences of the United States of America, 119 (25), ISSN:0027-8424, ISSN:1091-6490
- Published
- 2022
- Full Text
- View/download PDF
9. Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo
- Author
-
Kevin A. Wilkinson, Kirsty J. McMillan, Paul J. Banks, Ruth E. Carmichael, Yasuko Nakamura, Zafar I. Bashir, Peter J. Cullen, and Jeremy M. Henley
- Published
- 2022
10. TFEB controls retromer expression in response to nutrient availability
- Author
-
Peter J. Cullen, Alessia Calcagni, Andrea Ballabio, Rachel Curnock, Curnock, R., Calcagni, A., Ballabio, A., and Cullen, P. J.
- Subjects
Male ,Amino Acid Transport System A ,Retromer ,oncogenes ,Glutamine ,Vesicular Transport Proteins ,Mice ,VPS35 ,0302 clinical medicine ,Promoter Regions, Genetic ,genes ,VPS26A ,Research Articles ,transcription factor ,0303 health sciences ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Up-Regulation ,Cell biology ,glutamine ,sorting ,Signal Transduction ,Amino Acid Transport System ASC ,Endosome ,Endosomes ,Biology ,membrane transport proteins ,staarvation ,Large Neutral Amino Acid-Transporter 1 ,Minor Histocompatibility Antigens ,03 medical and health sciences ,nutrients ,Report ,Animals ,Humans ,Transcription factor ,030304 developmental biology ,amino acids ,Cell Membrane ,Promoter ,Nutrients ,Cell Biology ,Mice, Inbred C57BL ,HEK293 Cells ,mitf protein ,TFEB ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
The retromer complex is responsible for the endosomal sorting of numerous nutrient transporters. In this study, Curnock et al. show that in response to nutrient deprivation, retromer is transcriptionally regulated by TFEB and acts to support adaptive nutrient acquisition through endosomal recycling of the glutamine transporter, SNAT2 (SLC38A2)., Endosomal recycling maintains the cell surface abundance of nutrient transporters for nutrient uptake, but how the cell integrates nutrient availability with recycling is less well understood. Here, in studying the recycling of human glutamine transporters ASCT2 (SLC1A5), LAT1 (SLC7A5), SNAT1 (SLC38A1), and SNAT2 (SLC38A2), we establish that following amino acid restriction, the adaptive delivery of SNAT2 to the cell surface relies on retromer, a master conductor of endosomal recycling. Upon complete amino acid starvation or selective glutamine depletion, we establish that retromer expression is upregulated by transcription factor EB (TFEB) and other members of the MiTF/TFE family of transcription factors through association with CLEAR elements in the promoters of the retromer genes VPS35 and VPS26A. TFEB regulation of retromer expression therefore supports adaptive nutrient acquisition through endosomal recycling.
- Published
- 2019
11. Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins
- Author
-
Michael D. Healy, Joanna Sacharz, Kerrie E. McNally, Calum McConville, Vikas A. Tillu, Ryan J. Hall, Molly Chilton, Peter J. Cullen, Mehdi Mobli, Rajesh Ghai, David A. Stroud, and Brett M. Collins
- Subjects
Proteomics ,Structural Biology ,Amino Acid Sequence ,Endosomes ,Sorting Nexins ,Molecular Biology ,Protein Binding - Abstract
The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
- Published
- 2022
12. SNX27-Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling
- Author
-
Boris Simonetti, Qian Guo, Manuel Giménez-Andrés, Kai-En Chen, Edmund R. R. Moody, Ashley J. Evans, Mintu Chandra, Chris M. Danson, Tom A. Williams, Brett M. Collins, and Peter J. Cullen
- Subjects
Protein Transport ,General Immunology and Microbiology ,General Neuroscience ,Cell Membrane ,Animals ,macromolecular substances ,Endosomes ,General Agricultural and Biological Sciences ,Sorting Nexins ,General Biochemistry, Genetics and Molecular Biology - Abstract
Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.
- Published
- 2021
13. Mechanistic basis for SNX27-Retromer coupling to ESCPE-1 in promoting endosomal cargo recycling
- Author
-
Kai-En Chen, Edmund R. R. Moody, Peter J. Cullen, Ashley J. Evans, Boris Simonetti, Tom A. Williams, Manuel Gimenez-Andres, Brett M. Collins, Qian Guo, and Chris M. Danson
- Subjects
SNX27 ,Ezrin ,Retromer ,Radixin ,Cell adhesion molecule ,Endosome ,Chemistry ,Moesin ,Integral membrane protein ,Cell biology - 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.
- Published
- 2021
14. Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins
- Author
-
David A. Stroud, Rajesh Ghai, Michael D. Healy, Calum McConville, Molly Chilton, Ryan J. Hall, Joanna Sacharz, Brett M. Collins, Mehdi Mobli, Peter J. Cullen, and Kerrie E. McNally
- Subjects
Sorting nexin ,biology ,Protein family ,Chemistry ,PDZ domain ,Integrin ,Amyloid precursor protein ,biology.protein ,Transmembrane protein ,Proto-oncogene tyrosine-protein kinase Src ,LIM domain ,Cell biology - Abstract
The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/454387v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@137afd1org.highwire.dtl.DTLVardef@f7c91eorg.highwire.dtl.DTLVardef@1b4f2eborg.highwire.dtl.DTLVardef@b82f7c_HPS_FORMAT_FIGEXP M_FIG C_FIG
- Published
- 2021
15. Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2
- Author
-
Paul J Banks, Zafar I. Bashir, Philip A. Lewis, Brett M. Collins, Kirsty J McMillan, Peter J. Cullen, Ruth E. Carmichael, Jeremy M. Henley, Kevin A. Wilkinson, Thomas Clairfeuille, Kate J. Heesom, Francesca L N Hellel, and Ashley J. Evans
- Subjects
0301 basic medicine ,Proteomics ,SNX27 ,Long-Term Potentiation ,Excitotoxicity ,medicine.disease_cause ,Hippocampus ,Synaptic Transmission ,0302 clinical medicine ,Biology (General) ,AMPA receptors ,Integral membrane protein ,Sorting Nexins ,Neurons ,0303 health sciences ,Membrane Glycoproteins ,Chemistry ,General Neuroscience ,membrane trafficking ,Long-term potentiation ,General Medicine ,Cell biology ,Protein Transport ,Medicine ,Research Article ,Human ,QH301-705.5 ,Endosome ,Science ,Nerve Tissue Proteins ,AMPA receptor ,Endosomes ,Neurotransmission ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,medicine ,Animals ,Humans ,Receptors, AMPA ,030304 developmental biology ,Memory Disorders ,General Immunology and Microbiology ,Cell Biology ,Rats ,Sorting nexin ,030104 developmental biology ,LRFN2 ,Rat ,030217 neurology & neurosurgery ,Neuroscience - Abstract
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 have performed unbiased proteomics to identify new neuronal SNX27-dependent cargoes, and identified proteins linked to excitotoxicity (SLC1A3, SLC4A7, SLC6A11), epilepsy, intellectual disabilities and working memory deficits (KCNT2, ADAM22, KIDINS220, LRFN2). Focusing on the synaptic adhesion molecule leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2), we establish that SNX27 binds to LRFN2 and is responsible for regulating its endosomal sorting. LRFN2 associates with AMPA receptors and knockdown of LRFN2 phenocopies SNX27 depletion in decreasing surface expression of AMPA receptors, reducing synaptic activity and attenuating hippocampal long-term potentiation. Our evidence suggests that, in contrast to previous reports, SNX27 does not directly bind to AMPA receptors, and instead controls AMPA receptor-mediated synaptic transmission and plasticity indirectly through the endosomal sorting of LRFN2. Overall, our study provides new molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.
- Published
- 2021
16. Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins
- Author
-
Brett M. Collins, Greg J. Bashaw, Saroja Weeratunga, Madhavi Gorla, Blessy Paul, Kate J. Heesom, Karina Chaudhari, Florian Steinberg, Boris Simonetti, and Peter J. Cullen
- Subjects
Endosome ,Sorting Nexins ,Amino Acid Motifs ,Endosomes ,Semaphorins ,Endocytosis ,Article ,Receptor, IGF Type 2 ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,structural biology ,Animals ,Humans ,BAR domain ,030304 developmental biology ,0303 health sciences ,Chemistry ,membrane trafficking ,Membrane Proteins ,Cell Biology ,Transmembrane protein ,Cell biology ,Protein Transport ,A-site ,Drosophila melanogaster ,HEK293 Cells ,Structural biology ,endosomes ,030220 oncology & carcinogenesis ,Biogenesis ,HeLa Cells - Abstract
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX–BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.
- Published
- 2019
17. Proteomic Identification and Structural Basis for the Interaction between Sorting Nexin Snx17 and Pdlim Family Proteins
- Author
-
Joanna Sacharz, Ryan J. Hall, Mehdi Mobli, Brett M. Collins, Rajesh Ghai, Peter J. Cullen, Michael D. Healy, Calum McConville, David A. Stroud, Molly Chilton, and Kerrie E. McNally
- Subjects
History ,Polymers and Plastics ,biology ,Protein family ,Chemistry ,PDZ domain ,Integrin ,Proteomics ,Industrial and Manufacturing Engineering ,Transmembrane protein ,Cell biology ,Sorting nexin ,Amyloid precursor protein ,biology.protein ,Business and International Management ,LIM domain - Abstract
The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
- Published
- 2021
18. Sorting nexin 5 mediates virus-induced autophagy and immunity
- Author
-
Elizabeth R. Aguilera, Xuewu Zhang, Liwei Wang, Sandra L. Schmid, Nicholas T. Ktistakis, Bo Ci, Ramnik J. Xavier, John W. Schoggins, Julie K. Pfeiffer, Xiaonan Dong, Boris Simonetti, Madhura Bhave, Yuting Yang, R. Blake Richardson, Peter J. Cullen, Xiao Zang, Y. Zhao, Paul A. Gleeson, Guanghua Xiao, Li Yu, Yi Chun Kuo, Lin Zhong, Yang Xie, Zhongju Zou, Beth Levine, and Rui Zhong
- Subjects
Male ,Small interfering RNA ,Endosome ,Vesicular Transport Proteins ,Lipid kinase activity ,Autophagy-Related Proteins ,Endosomes ,In Vitro Techniques ,Biology ,Cell Line ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Interferon ,Autophagy ,medicine ,Animals ,Humans ,RNA, Small Interfering ,Sorting Nexins ,PI3K/AKT/mTOR pathway ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Class III Phosphatidylinositol 3-Kinases ,Cell biology ,Mice, Inbred C57BL ,Sorting nexin ,Viruses ,Beclin-1 ,Female ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.
- Published
- 2020
19. Neuropilin-1 is a host factor for SARS-CoV-2 infection
- Author
-
Deborah K. Shoemark, James L. Daly, Maia Kavanagh Williamson, Peter Horvath, David A. Matthews, Lorena Simón-Gracia, Yohei Yamauchi, Carlos Antón-Plágaro, Reka Hollandi, Urs F. Greber, Boris Simonetti, Ari Helenius, Julian A. Hiscox, Tambet Teesalu, Brett M. Collins, Andrew D. Davidson, Richard B. Sessions, Peter J. Cullen, Kai-En Chen, Michael Bauer, Katja Klein, University of Zurich, and Simonetti, Boris
- Subjects
medicine.medical_treatment ,viruses ,UNCOVER ,BrisSynBio ,Plasma protein binding ,Peptidyl-Dipeptidase A ,Multidisciplinary / COVID ,Article ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Neuropilin 1 ,medicine ,Humans ,Furin ,030304 developmental biology ,Host factor ,chemistry.chemical_classification ,Infectivity ,0303 health sciences ,1000 Multidisciplinary ,Protease ,Multidisciplinary ,biology ,SARS-CoV-2 ,Bristol BioDesign Institute ,COVID-19 ,Covid19 ,Virus Internalization ,Virology ,10124 Institute of Molecular Life Sciences ,Neuropilin-1 ,3. Good health ,chemistry ,biology.protein ,570 Life sciences ,Glycoprotein ,030217 neurology & neurosurgery - Abstract
Another host factor for SARS-CoV-2 Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science , this issue p. 856 , p. 861 ; see also p. 765
- Published
- 2020
- Full Text
- View/download PDF
20. Author response: Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2
- Author
-
Kirsty J McMillan, Paul J Banks, Francesca LN Hellel, Ruth E Carmichael, Thomas Clairfeuille, Ashley J Evans, Kate J Heesom, Philip Lewis, Brett M Collins, Zafar I Bashir, Jeremy M Henley, Kevin A Wilkinson, and Peter J Cullen
- Published
- 2020
21. Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting
- Author
-
Anis N. K. Anuar, Boris Simonetti, James L. Daly, Peter J. Cullen, and Ashley J. Evans
- Subjects
Retromer ,Endosome ,Vesicular Transport Proteins ,Regulator ,Endosomes ,Biology ,03 medical and health sciences ,VPS35 ,ESCPE-1 ,0302 clinical medicine ,Humans ,Sorting Nexins ,VPS26A ,030304 developmental biology ,0303 health sciences ,Knocksideways ,Insulin-like growth factor 2 receptor ,Sorting ,Cell Biology ,Cell biology ,Protein Transport ,VPS29 ,CI-MPR ,SNX5 ,GLUT1 ,030217 neurology & neurosurgery ,Research Article ,HeLa Cells ,trans-Golgi Network - Abstract
Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex – a key regulator of retrograde CI-MPR sorting – revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting., Summary: Retromer, a master controller of endosomal cargo sorting, is deregulated in neurodegenerative disease. Here, we develop and apply a retromer knocksideways methodology to quantify endosomal cargo sorting upon acute perturbation.
- Published
- 2020
22. Neuropilin-1 is a host factor for SARS-CoV-2 infection
- Author
-
Urs F. Greber, Ari Helenius, James L. Daly, David A. Matthews, Andrew D. Davidson, Katja Klein, Richard B. Sessions, Michael Bauer, Deborah K. Shoemark, Carlos Antón-Plágaro, Peter Horvath, Maia Kavanagh Williamson, Yohei Yamauchi, Lorena Simón-Gracia, Julian A. Hiscox, Tambet Teesalu, Boris Simonetti, Reka Hollandi, and Peter J. Cullen
- Subjects
0303 health sciences ,biology ,Immunoprecipitation ,Chemistry ,medicine.disease_cause ,Virus ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Neuropilin 1 ,medicine ,biology.protein ,Sequence motif ,Protein precursor ,Furin ,030304 developmental biology ,Coronavirus ,Host factor - Abstract
SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(3,4). For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors5. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.
- Published
- 2020
23. Mammalian copper homeostasis requires retromer-dependent recycling of the high-affinity copper transporter 1
- Author
-
Rachel Curnock and Peter J. Cullen
- Subjects
Retromer ,Endosome ,ATPase ,ATP7A ,chemistry.chemical_element ,03 medical and health sciences ,0302 clinical medicine ,Extracellular ,Animals ,Homeostasis ,Metal ion homeostasis ,Cation Transport Proteins ,030304 developmental biology ,Copper Transporter 1 ,0303 health sciences ,biology ,Endosomal recycling ,fungi ,CTR1 ,Transporter ,Cell Biology ,Copper ,Cell biology ,chemistry ,biology.protein ,Cisplatin ,030217 neurology & neurosurgery ,Research Article - Abstract
The concentration of essential micronutrients, such as copper (used here to describe both Cu+ and Cu2+), within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. Retromer-mediated sorting and recycling of nutrient transporters within the endo-lysosomal network is an essential process in regulating nutrient balance. Cellular copper homeostasis is regulated primarily by two transporters: the copper influx transporter copper transporter 1 (CTR1; also known as SLC31A1), which controls the uptake of copper, and the copper-extruding ATPase ATP7A, a recognised retromer cargo. Here, we show that in response to fluctuating extracellular copper, retromer controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and, in response to decreased extracellular copper, retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in the trafficking of platinum. The efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug cisplatin., Summary: CTR1 (SLC31A1) is the only known mammalian importer of copper. We show that CTR1 is a retromer complex cargo protein, and that retromer is required for cellular sensitivity to extracellular copper.
- Published
- 2020
24. Mammalian copper homeostasis requires retromer-dependent recycling of the high-affinity copper transporter 1 (CTR1/SLC31A1)
- Author
-
Rachel Curnock and Peter J. Cullen
- Subjects
Retromer ,biology ,Endosome ,Chemistry ,ATPase ,fungi ,ATP7A ,chemistry.chemical_element ,Copper ,Cell biology ,Retromer complex ,biology.protein ,Extracellular ,Integral membrane protein - Abstract
The mammalian cell surface is decorated with a plethora of integral membrane proteins including those required for the transport of micronutrients, such as copper, which are essential to cellular health. The concentration of micronutrients within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. The sorting and recycling of nutrients transporters within the endo-lysosomal network is recognised as an essential process in regulating nutrient balance. The evolutionarily conserved endosomal sorting complex, retromer, coordinates integral membrane protein recognition and retrieval. Cellular copper homeostasis is regulated primarily by two transporters: the major copper influx transporter copper transporter 1 (CTR1/SLC31A1), which controls the uptake of copper from the extracellular environment and is essential for early embryonic development, and the established retromer cargo, the copper-transporting ATPase, ATP7A. Here, we show that in response to fluctuating extracellular copper the retromer complex controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and in response to decreased extracellular copper retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in platinum uptake. Significantly, the efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug, cisplatin.
- Published
- 2020
25. A heterodimeric SNX4:SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly
- Author
-
Virginie M S Betin, Jon D. Lane, Boris Simonetti, Naomi Attar, Peter J. Cullen, Colin J. Traer, and Zuriñe Antón
- Subjects
Autophagosome ,autophagy ,Endosome ,Sorting Nexins ,Endocytic cycle ,Lipid-anchored protein ,Endosomes ,Biology ,sorting nexin ,Sorting nexin ,03 medical and health sciences ,0302 clinical medicine ,Autophagy ,Animals ,Endomembrane system ,ATG9A ,030304 developmental biology ,0303 health sciences ,Chemistry ,030302 biochemistry & molecular biology ,Peripheral membrane protein ,Autophagosomes ,Cell Biology ,Cell biology ,Protein Transport ,endosomes ,SNX30 ,Autophagosome assembly ,SNX4 ,SNX7 ,030217 neurology & neurosurgery ,Biogenesis ,Research Article - Abstract
The sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4–SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4–SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy., Summary: A heterodimeric SNX4–SNX7 SNX-BAR complex regulates mammalian autophagosome assembly through the control of ATG9 trafficking.
- Published
- 2020
26. Acute perturbation of retromer and ESCPE-1 leads to functionally distinct and temporally resolved defects in endosomal cargo sorting
- Author
-
James L. Daly, Peter J. Cullen, Ashley J. Evans, Anis N. K. Anuar, and Boris Simonetti
- Subjects
0303 health sciences ,Retromer ,Endosome ,Chemistry ,Cell ,Regulator ,Cell biology ,03 medical and health sciences ,VPS35 ,0302 clinical medicine ,medicine.anatomical_structure ,VPS29 ,medicine ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The human retromer is a stable heterotrimer of VPS35, VPS29 and VPS26 whose principal role is to orchestrate the endosomal retrieval of hundreds of internalised cargo and promote their recycling to the cell surface; a prototypical cargo being the glucose transporter GLUT1. Retromer’s role in a distinct endosomal retrieval pathway, the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network (TGN), remains controversial. Here we have developed and applied knocksideways to acutely inactivate retromer and by visualising the sorting of endogenous GLUT1 and CI-MPR provide insight into the temporal dynamics of endosomal cargo sorting in HeLa and H4 human neuroglioma cells. While retromer knocksideways led to the development of time-resolved defects in cell surface sorting of GLUT1 we failed to observe defects in the sorting of the CI-MPR. In contrast knocksideways of ESCPE-1, a key regulator of retrograde CI-MPR sorting, resulted in a time-resolved defect in CI-MPR sorting. Together these data provide independent evidence consistent with a comparatively limited role for retromer in ESCPE-1 dependent CI-MPR retrograde sorting in HeLa and H4 human neuroglioma cells.
- Published
- 2019
- Full Text
- View/download PDF
27. Endoplasmic Reticulum–Endosome Contact Sites: Specialized Interfaces for Orchestrating Endosomal Tubule Fission?
- Author
-
James L. Daly and Peter J. Cullen
- Subjects
0301 basic medicine ,Binding Sites ,Endosome ,Chemistry ,Fission ,Endoplasmic reticulum ,Membrane Proteins ,Endosomes ,Endoplasmic Reticulum ,Models, Biological ,Biochemistry ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Tubule ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Binding site ,030217 neurology & neurosurgery - Abstract
[No Abstract]
- Published
- 2018
28. Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR
- Author
-
Peter J. Cullen, Boris Simonetti, Kate J. Heesom, and Chris M. Danson
- Subjects
0301 basic medicine ,Retromer ,Endosome ,Sorting Nexins ,Cell Biology ,Biology ,Transmembrane protein ,Transport protein ,Cell biology ,03 medical and health sciences ,Sorting nexin ,030104 developmental biology ,VPS29 ,Amphiphysin - Abstract
Endosomal recycling of transmembrane proteins requires sequence-dependent recognition of motifs present within their intracellular cytosolic domains. In this study, we have reexamined the role of retromer in the sequence-dependent endosome-to–trans-Golgi network (TGN) transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Although the knockdown or knockout of retromer does not perturb CI-MPR transport, the targeting of the retromer-linked sorting nexin (SNX)–Bin, Amphiphysin, and Rvs (BAR) proteins leads to a pronounced defect in CI-MPR endosome-to-TGN transport. The retromer-linked SNX-BAR proteins comprise heterodimeric combinations of SNX1 or SNX2 with SNX5 or SNX6 and serve to regulate the biogenesis of tubular endosomal sorting profiles. We establish that SNX5 and SNX6 associate with the CI-MPR through recognition of a specific WLM endosome-to-TGN sorting motif. From validating the CI-MPR dependency of SNX1/2–SNX5/6 tubular profile formation, we provide a mechanism for coupling sequence-dependent cargo recognition with the biogenesis of tubular profiles required for endosome-to-TGN transport. Therefore, the data presented in this study reappraise retromer’s role in CI-MPR transport.
- Published
- 2017
29. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling
- Author
-
Lindsey L. Morris, Lawrence Banks, David Pim, Paul F. Langton, Amika Singla, Neil D. Pearson, Rebecca A. Faulkner, Kerrie E. McNally, Ezra Burstein, Chris M. Danson, Richard B. Sessions, Matthew Gallon, Rajesh Ghai, Brittany L. Overlee, Brett M. Collins, Heike Nägele, Imre Berger, Daniel D. Billadeau, Peter J. Cullen, Kate J. Heesom, and Florian Steinberg
- Subjects
Models, Molecular ,Proteomics ,0301 basic medicine ,Cell biology ,Multiprotein complex ,Retromer ,Endosome ,Vesicular Transport Proteins ,Membrane trafficking ,Endosomes ,Transfection ,Endocytosis ,Animals, Genetically Modified ,WASH complex ,Structure-Activity Relationship ,03 medical and health sciences ,Animals ,Drosophila Proteins ,Humans ,Protein Interaction Domains and Motifs ,Sorting Nexins ,Integral membrane protein ,Protein Stability ,Chemistry ,Cell Membrane ,Intracellular Signaling Peptides and Proteins ,Proteins ,Cell Biology ,Neoplasm Proteins ,Kinetics ,Protein Transport ,Sorting nexin ,Drosophila melanogaster ,HEK293 Cells ,030104 developmental biology ,VPS29 ,Multiprotein Complexes ,Proteolysis ,RNA Interference ,HeLa Cells ,Protein Binding - Abstract
Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.
- Published
- 2017
30. Editorial overview: Membrane trafficking
- Author
-
Akihiko Nakano and Peter J. Cullen
- Subjects
Membrane ,Cell Biology ,Biology ,Cell biology - Abstract
[No Abstract]
- Published
- 2019
31. 3. Money Laundering: The European Community Directive
- Author
-
Peter J. Cullen
- Published
- 2019
32. Endosomal Sorting:Architecture of the Retromer Coat
- Author
-
Boris Simonetti and Peter J. Cullen
- Subjects
0301 basic medicine ,Multiprotein complex ,Retromer ,Endosome ,Sorting Nexins ,Sorting ,Master regulator ,macromolecular substances ,Biology ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Transport protein ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Vesicular Transport Proteins ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery - Abstract
Retromer is a master regulator of endosomal cargo sorting. Using cryo-EM, a new study now reveals how, in yeast, this multiprotein complex is assembled on tubular membranes to form a coat complex that orchestrates the process of tubular-based cargo sorting.
- Published
- 2018
33. To degrade or not to degrade:mechanisms and significance of endocytic recycling
- Author
-
Peter J. Cullen and Florian Steinberg
- Subjects
0301 basic medicine ,Cell adhesion molecule ,Endosome ,Chemistry ,Cell ,Cell Membrane ,Endocytic recycling ,Membrane Proteins ,Cell Biology ,Endosomes ,Endocytosis ,Cell biology ,03 medical and health sciences ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,medicine ,Animals ,Humans ,Molecular Biology ,Integral membrane protein ,Function (biology) ,Organism ,Ion channel - Abstract
Newly endocytosed integral cell surface proteins are typically either directed for degradation or subjected to recycling back to the plasma membrane. The sorting of integral cell surface proteins, including signalling receptors, nutrient transporters, ion channels, adhesion molecules and polarity markers, within the endolysosomal network for recycling is increasingly recognized as an essential feature in regulating the complexities of physiology at the cell, tissue and organism levels. Historically, endocytic recycling has been regarded as a relatively passive process, where the majority of internalized integral proteins are recycled via a nonspecific sequence-independent 'bulk membrane flow' pathway. Recent work has increasingly challenged this view. The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process.
- Published
- 2018
34. Parkinson’s disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes
- Author
-
Alan L Whone, Hisashi Fujioka, Maeve A. Caldwell, Xiongwei Zhu, Xinglong Wang, Jun Liu, Wenzhang Wang, Peter J. Cullen, and Charles L. Hoppel
- Subjects
Dynamins ,0301 basic medicine ,Male ,Parkinson's disease ,Blotting, Western ,Vesicular Transport Proteins ,Fluorescent Antibody Technique ,Substantia nigra ,In Vitro Techniques ,Biology ,Time-Lapse Imaging ,Mitochondrial Dynamics ,General Biochemistry, Genetics and Molecular Biology ,Article ,GTP Phosphohydrolases ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,VPS35 ,Microscopy, Electron, Transmission ,Cellular neuroscience ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Microscopy, Immunoelectron ,Aged ,Aged, 80 and over ,Neurons ,Neurodegeneration ,Parkinson Disease ,General Medicine ,medicine.disease ,Rats ,Cell biology ,Mitochondria ,Substantia Nigra ,Retromer complex ,Oxidative Stress ,030104 developmental biology ,Gene Knockdown Techniques ,DNAJA3 ,Mitochondrial fission ,Female ,Microtubule-Associated Proteins ,Fluorescence Recovery After Photobleaching - Abstract
Mutations in VPS35 that are associated with Parkinson's disease increase the interaction of VPS35 with mitochondrial DLP1, leading to removal of the DLP1 complexes and mitochondrial fragmentation. Structural and functional mitochondrial impairments caused by mutant VPS35 are observed in vitro using cultured neurons and fibroblasts from individuals with PD and in vivo in mouse substantia nigra neurons, where they induce neurodegeneration. Mitochondrial dysfunction represents a critical step during the pathogenesis of Parkinson's disease (PD), and increasing evidence suggests abnormal mitochondrial dynamics and quality control as important underlying mechanisms. The VPS35 gene, which encodes a key component of the membrane protein–recycling retromer complex, is the third autosomal-dominant gene associated with PD. However, how VPS35 mutations lead to neurodegeneration remains unclear. Here we demonstrate that PD-associated VPS35 mutations caused mitochondrial fragmentation and cell death in cultured neurons in vitro, in mouse substantia nigra neurons in vivo and in human fibroblasts from an individual with PD who has the VPS35D620N mutation. VPS35-induced mitochondrial deficits and neuronal dysfunction could be prevented by inhibition of mitochondrial fission. VPS35 mutants showed increased interaction with dynamin-like protein (DLP) 1, which enhanced turnover of the mitochondrial DLP1 complexes via the mitochondria-derived vesicle–dependent trafficking of the complexes to lysosomes for degradation. Notably, oxidative stress increased the VPS35-DLP1 interaction, which we also found to be increased in the brains of sporadic PD cases. These results revealed a novel cellular mechanism for the involvement of VPS35 in mitochondrial fission, dysregulation of which is probably involved in the pathogenesis of familial, and possibly sporadic, PD.
- Published
- 2015
35. Endosomal Retrieval of Cargo:Retromer Is Not Alone
- Author
-
Peter J. Cullen and Kerrie E. McNally
- Subjects
0301 basic medicine ,Retromer ,Endosome ,sorting nexin (SNX) ,WASH complex ,Cellular homeostasis ,Cell Biology ,Biology ,medicine.disease_cause ,Transmembrane protein ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Membrane protein ,endosomes ,CCC complex ,Protein targeting ,medicine ,retriever ,Receptor ,retromer ,Integral membrane protein ,030217 neurology & neurosurgery - Abstract
Endosomes are major protein sorting stations in cells. Endosomally localised multi-protein complexes sort integral proteins, including signaling receptors, nutrient transporters, adhesion molecules, and lysosomal hydrolase receptors, for lysosomal degradation or conversely for retrieval and subsequent recycling to various membrane compartments. Correct endosomal sorting of these proteins is essential for maintaining cellular homeostasis, with defects in endosomal sorting implicated in various human pathologies including neurodegenerative disorders. Retromer, an ancient multi-protein complex, is essential for the retrieval and recycling of hundreds of transmembrane proteins. While retromer is a major player in endosomal retrieval and recycling, several studies have recently identified retrieval mechanisms that are independent of retromer. Here, we review endosomal retrieval complexes, with a focus on recently discovered retromer-independent mechanisms.
- Published
- 2018
36. Sorting nexin-21 is a scaffold for the endosomal recruitment of huntingtin
- Author
-
Peter J. Cullen, Neil D. Pearson, Chris M. Danson, and Kate J. Heesom
- Subjects
0301 basic medicine ,Huntingtin Protein ,education.field_of_study ,Huntingtin ,Retromer ,Endosome ,Sorting Nexins ,Population ,Endosomes ,Cell Biology ,Biology ,Septin ,Interactome ,Cell Line ,Cell biology ,Protein Transport ,03 medical and health sciences ,Sorting nexin ,030104 developmental biology ,Humans ,Protein Interaction Domains and Motifs ,education ,Septins ,Protein Binding - 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.
- Published
- 2018
37. Retromer Controls Planar Polarity Protein Levels and Asymmetric Localization at Intercellular Junctions
- Author
-
Helen, Strutt, Paul F, Langton, Neil, Pearson, Kirsty J, McMillan, David, Strutt, and Peter J, Cullen
- Subjects
Male ,Pupa ,Cell Polarity ,planar cell polarity ,Endosomes ,Article ,Animals, Genetically Modified ,PCP ,Strabismus ,Protein Transport ,sorting nexin 27 ,Drosophila melanogaster ,Intercellular Junctions ,WASH ,Flamingo ,Animals ,Drosophila Proteins ,Female ,Drosophila ,endosome recycling ,retromer - Abstract
Summary 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, 2, 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, 5, 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., Highlights • Retromer regulates levels and asymmetry of Flamingo and Strabismus in the pupal wing • Retromer regulates Flamingo and Strabismus levels independently of the Wash complex • The cargo adaptor Snx27 interacts directly with the PDZ-binding motif of Flamingo • Loss of Snx27 disrupts Flamingo trafficking, Planar cell polarity—the organization of cells within a two-dimensional plane—is an important feature in tissue organization. Strutt et al. show that, in the Drosophila wing, endosomal recycling through retromer and its cargo adaptor Snx27 mediates the cell-surface localization of two key planar polarity proteins, Flamingo and Strabismus.
- Published
- 2018
38. Actin-dependent endosomal receptor recycling
- Author
-
Peter J. Cullen and Boris Simonetti
- Subjects
Receptor recycling ,Retromer ,Endosome ,Sorting Nexins ,Vesicular Transport Proteins ,macromolecular substances ,Endosomes ,Biology ,WASH complex ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Humans ,030304 developmental biology ,0303 health sciences ,Membrane Proteins ,Cell Biology ,Transmembrane protein ,Actins ,Cell biology ,Transport protein ,Protein Transport ,Multiprotein Complexes ,Carrier Proteins ,Lysosomes ,Cortactin ,030217 neurology & neurosurgery ,Biogenesis ,Wiskott-Aldrich Syndrome Protein - Abstract
Endosomes constitute major sorting compartments within the cell. There, a myriad of transmembrane proteins (cargoes) are delivered to the lysosome for degradation or retrieved from this fate and recycled through tubulo-vesicular transport carriers to different cellular destinations. Retrieval and recycling are orchestrated by multi-protein assemblies that include retromer and retriever, sorting nexins, and the Arp2/3 activating WASH complex. Fine-tuned control of actin polymerization on endosomes is fundamental for the retrieval and recycling of cargoes. Recent advances in the field have highlighted several roles that actin plays in this process including the binding to cargoes, stabilization of endosomal subdomains, generation of the remodeling forces required for the biogenesis of cargo-enriched transport carriers and short-range motility of the transport carriers.
- Published
- 2018
39. Author response: Structural insights into the architecture and membrane interactions of the conserved COMMD proteins
- Author
-
Molly Chilton, Mintu Chandra, Vikas A. Tillu, Peter J. Cullen, Manuela K. Hospenthal, Ryan J. Hall, Dion J. Celligoi, Fiona J. McDonald, J. Shaun Lott, Rajesh Ghai, Brett M. Collins, Kai-En Chen, and Michael D. Healy
- Subjects
Membrane ,Chemistry ,Computational biology ,Architecture - Published
- 2018
40. Retromer associates with the cytoplasmic amino-terminus of polycystin-2
- Author
-
Chris M. Danson, Matthew Gallon, Jing Zhou, Peter J. Cullen, Frances C. Tilley, and Chong Luo
- Subjects
0301 basic medicine ,endocrine system ,TRPP Cation Channels ,Retromer ,Endosome ,Amino Acid Motifs ,Population ,Autosomal dominant polycystic kidney disease ,Endosomes ,Biology ,urologic and male genital diseases ,Gene Knockout Techniques ,Mice ,03 medical and health sciences ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,education ,Integral membrane protein ,education.field_of_study ,PKD1 ,urogenital system ,Cell Biology ,Polycystic Kidney, Autosomal Dominant ,medicine.disease ,female genital diseases and pregnancy complications ,3. Good health ,Cell biology ,Vesicular transport protein ,HEK293 Cells ,030104 developmental biology ,Polycystin 2 ,Multiprotein Complexes ,HeLa Cells ,Research Article - Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic human disease, with around 12.5 million people affected worldwide. ADPKD results from mutations in either PKD1 or PKD2, which encode the atypical G-protein coupled receptor polycystin-1 (PC1) and the transient receptor potential channel polycystin-2 (PC2), respectively. Although altered intracellular trafficking of PC1 and PC2 is an underlying feature of ADPKD, the mechanisms which govern vesicular transport of the polycystins through the biosynthetic and endosomal membrane networks remain to be fully elucidated. Here, we describe an interaction between PC2 and retromer, a master controller for the sorting of integral membrane proteins through the endo-lysosomal network. We show that association of PC2 with retromer occurs via a region in the PC2 cytoplasmic amino-terminal domain, independently of the retromer-binding Wiskott-Aldrich syndrome and scar homologue (WASH) complex. Based on observations that retromer preferentially interacts with a trafficking population of PC2, and that ciliary levels of PC1 are reduced upon mutation of key residues required for retromer association in PC2, our data are consistent with the identification of PC2 as a retromer cargo protein.This article has an associated First Person interview with the first author of the paper.
- Published
- 2018
41. Retromer and sorting nexins in endosomal sorting
- Author
-
Matthew Gallon and Peter J. Cullen
- Subjects
Models, Molecular ,Retromer ,Endosome ,Sorting Nexins ,Cell Membrane ,Vesicular Transport Proteins ,Endosomes ,Protein degradation ,Biology ,Biochemistry ,Endocytosis ,Transport protein ,Cell biology ,Retromer complex ,Protein Transport ,Sorting nexin ,Multiprotein Complexes ,Animals ,Humans ,Endomembrane system ,Protein Structure, Quaternary ,trans-Golgi Network - Abstract
The evolutionarily conserved endosomal retromer complex rescues transmembrane proteins from the lysosomal degradative pathway and facilitates their recycling to other cellular compartments. Retromer functions in conjunction with numerous associated proteins, including select members of the sorting nexin (SNX) family. In the present article, we review the molecular architecture and cellular roles of retromer and its various functional partners. The endosomal network is a crucial hub in the trafficking of proteins through the cellular endomembrane system. Transmembrane proteins, here termed cargos, enter endosomes by endocytosis from the plasma membrane or by trafficking from the trans-Golgi network (TGN). Endosomal cargo proteins face one of the two fates: retention in the endosome, leading ultimately to lysosomal degradation or export from the endosome for reuse (‘recycling’). The balance of protein degradation and recycling is crucial to cellular homoeostasis; inappropriate sorting of proteins to either fate leads to cellular dysfunction. Retromer is an endosome-membrane-associated protein complex central to the recycling of many cargo proteins from endosomes, both to the TGN and the plasma membrane (and other specialized compartments, e.g. lysosome-related organelles). Retromer function is reliant on a number of proteins from the SNX family. In the present article, we discuss this inter-relationship and how defects in retromer function are increasingly being linked with human disease.
- Published
- 2015
42. Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50
- Author
-
Ana Jimenez Orgaz, Arunas Kvainickas, Kate J. Heesom, Jörn Dengjel, Florian Steinberg, Heike Nägele, Britta Diedrich, and Peter J. Cullen
- Subjects
Amino Acid Transport System ASC ,Proteomics ,0301 basic medicine ,Integrins ,SNX27 ,Retromer ,Endosome ,Amino Acid Motifs ,Vesicular Transport Proteins ,Endocytic recycling ,Membrane trafficking ,Endosomes ,Biology ,Mass Spectrometry ,WASH complex ,03 medical and health sciences ,VPS35 ,Phosphoprotein Phosphatases ,Humans ,Protein Interaction Maps ,Sorting Nexins ,Integral membrane protein ,Glucose Transporter Type 1 ,Cell Membrane ,Microfilament Proteins ,Transferrin ,Biological Transport ,Cell Biology ,Endocytosis ,Transmembrane protein ,Cell biology ,ErbB Receptors ,HEK293 Cells ,030104 developmental biology ,Gene Knockdown Techniques ,Proteolysis ,Research Article ,HeLa Cells ,Protein Binding - 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 Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's 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.
- Published
- 2017
43. The emerging role of retromer in neuroprotection
- Author
-
Kirsty J, McMillan, Hendrick C, Korswagen, and Peter J, Cullen
- Subjects
Protein Transport ,Eukaryotic Cells ,Endosomal Sorting Complexes Required for Transport ,Proteolysis ,Animals ,Humans ,Parkinson Disease ,Neuroprotection ,Article ,Cell-Matrix Junctions - Abstract
Highlights • In the endosomal network retromer retrieves cargo away from the degradative pathway. • Retromer dysfunction has been implicated in Parkinson’s disease through different mechanisms. • These include changes in protein association, protein degradation and mitochondria quality control., Efficient sorting and transportation of integral membrane proteins, such as ion channels, nutrient transporters, signalling receptors, cell–cell and cell–matrix adhesion molecules is essential for the function of cellular organelles and hence organism development and physiology. Retromer is a master controller of integral membrane protein sorting and transport through one of the major sorting station within eukaryotic cells, the endosomal network. Subtle de-regulation of retromer is an emerging theme in the pathoetiology of Parkinson’s disease. Here we summarise recent advances in defining the neuroprotective role of retromer and how its de-regulation may contribute to Parkinson’s disease by interfering with: lysosomal health and protein degradation, association with accessory proteins including the WASH complex and mitochondrial health.
- Published
- 2016
44. A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport
- Author
-
Jeremy M. Tavaré, Elaine C. Thomas, Florian Steinberg, Amanda J. Bell, Matthew Gallon, Kate J. Heesom, Mark O. Winfield, and Peter J. Cullen
- Subjects
Proteomics ,Protein Folding ,SNX27 ,Retromer ,Endosome ,Metal ion transport ,Blotting, Western ,Vesicular Transport Proteins ,PDZ Domains ,Biology ,Article ,Receptor, Platelet-Derived Growth Factor beta ,VPS35 ,Protein Interaction Mapping ,Humans ,RNA, Small Interfering ,Cation Transport Proteins ,Sorting Nexins ,Adenosine Triphosphatases ,Glucose Transporter Type 1 ,Ion Transport ,Computational Biology ,Cell Biology ,Culture Media ,Cell biology ,Retromer complex ,Protein Transport ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,Sorting nexin ,Glucose ,Copper-Transporting ATPases ,VPS29 ,Isotope Labeling ,Multiprotein Complexes ,Proteolysis ,Lysosomes ,HeLa Cells - Abstract
The PDZ-domain-containing sorting nexin 27 (SNX27) promotes recycling of internalized transmembrane proteins from endosomes to the plasma membrane by linking PDZ-dependent cargo recognition to retromer-mediated transport. Here, we employed quantitative proteomics of the SNX27 interactome and quantification of the surface proteome of SNX27- and retromer-suppressed cells to dissect the assembly of the SNX27 complex and provide an unbiased global view of SNX27-mediated sorting. Over 100 cell surface proteins, many of which interact with SNX27, including the glucose transporter GLUT1, the Menkes disease copper transporter ATP7A, various zinc and amino acid transporters, and numerous signalling receptors, require SNX27-retromer to prevent lysosomal degradation and maintain surface levels. Furthermore, we establish that direct interaction of the SNX27 PDZ domain with the retromer subunit VPS26 is necessary and sufficient to prevent lysosomal entry of SNX27 cargo. Our data identify the SNX27-retromer as a major endosomal recycling hub required to maintain cellular nutrient homeostasis.
- Published
- 2013
45. Atypical parkinsonism-associated retromer mutant alters endosomal sorting of specific cargo proteins
- Author
-
Frances C. Tilley, Chris M. Danson, Peter J. Cullen, Kate J. Heesom, Ian J. McGough, Adam P. Jellett, Matthew Gallon, Kirsty J McMillan, Kevin A. Wilkinson, Brett M. Collins, and Thomas Clairfeuille
- Subjects
0301 basic medicine ,SNX27 ,Retromer ,Endosome ,Sorting Nexins ,Endosomes ,Biology ,Cell Line ,03 medical and health sciences ,VPS35 ,Parkinsonian Disorders ,Report ,Protein Interaction Mapping ,Humans ,VPS26A ,Research Articles ,Cell Biology ,Cell biology ,Retromer complex ,Protein Subunits ,Protein Transport ,030104 developmental biology ,VPS29 ,Mutation - Abstract
Mutations in the retromer complex, which is involved in sorting integral membrane proteins from endosomes to cellular compartments, are associated with atypical parkinsonism, but how these mutations affect retromer function remains unclear. Through a quantitative proteomic analysis of the retromer interactome, McMillan et al. reveal a new mechanism for perturbed endosomal sorting in parkinsonism., 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.
- Published
- 2016
46. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules
- Author
-
James H. Hurley, Vikram K. Bhatia, Daniel P. Kloer, Peter J. Cullen, Sven R. Carlsson, Dimitrios Stamou, Colin J. Traer, Richard B. Sessions, and Jan R.T. van Weering
- Subjects
0303 health sciences ,genetic structures ,General Immunology and Microbiology ,Retromer ,Endosome ,General Neuroscience ,Sorting Nexins ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Transport protein ,03 medical and health sciences ,VPS35 ,Sorting nexin ,0302 clinical medicine ,Amphiphysin ,BAR domain ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX-BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX-BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 A structure of a SNX1-BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective ‘tip–loop' interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX-BAR-decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.
- Published
- 2012
47. SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways
- Author
-
Mark D. Bass, Peter J. Cullen, Kate J. Heesom, and Florian Steinberg
- Subjects
Proteomics ,SNX27 ,Integrin beta Chains ,Sorting Nexins ,Integrin ,Vesicular Transport Proteins ,Biological Transport, Active ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Report ,Cell Line, Tumor ,Humans ,RNA, Small Interfering ,Research Articles ,030304 developmental biology ,0303 health sciences ,Integrin beta1 ,HEK 293 cells ,Cell Biology ,Transmembrane protein ,Transport protein ,Cell biology ,Retromer complex ,Protein Transport ,HEK293 Cells ,Cytoplasm ,biology.protein ,RNA Interference ,Lysosomes ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Retrieval of β integrins from the lysosomal degradation pathway mediated by sorting nexin-17 is important for integrin recycling and regulation of cell migration., The FERM-like domain–containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture–based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.
- Published
- 2012
48. Phosphoinositides and the regulation of tubular-based endosomal sorting
- Author
-
Peter J. Cullen
- Subjects
Saccharomyces cerevisiae Proteins ,Endosome ,Cell Membrane ,Molecular Conformation ,Vesicular Transport Proteins ,Endosomes ,Saccharomyces cerevisiae ,Biology ,Phosphatidylinositols ,Biochemistry ,Cell function ,Molecular conformation ,Cell biology ,chemistry.chemical_compound ,chemistry ,Terminology as Topic ,Animals ,Humans ,Inositol ,Lysosomes ,Function (biology) - Abstract
From the pioneering work of Mabel and Lowell Hokin in the 1950s, the biology of this specific isomer of hexahydroxycyclohexane and its phosphorylated derivatives, in the form of inositol phosphates and phosphoinositides, has expanded to fill virtually every corner of cell biology, whole-organism physiology and development. In the present paper, I give a personal view of the role played by phosphoinositides in regulating the function of the endosomal network, and, in so doing, highlight some of the basic properties through which phosphoinositides regulate cell function.
- Published
- 2011
49. SNX–BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting
- Author
-
Peter J. Cullen, Jan R.T. van Weering, and Paul Verkade
- Subjects
Models, Molecular ,Retromer ,Protein Conformation ,Endosome ,Sorting Nexins ,Endocytic cycle ,Dynein ,Vesicular Transport Proteins ,Endosomes ,Biology ,Phosphatidylinositols ,Article ,Animals ,Humans ,Protein Isoforms ,BAR domain ,Phylogeny ,Molecular Motor Proteins ,Cell Membrane ,Biological Transport ,Cell Biology ,Clathrin ,Endocytosis ,Cell biology ,Sorting nexin ,Carrier Proteins ,trans-Golgi Network ,Developmental Biology - Abstract
The endocytic network is morphologically characterized by a wide variety of membrane bound compartments that are able to undergo dynamic re-modeling through tubular and vesicular structures. The precise molecular mechanisms governing such re-modeling, and the events that co-ordinated this with the major role of the endocytic network, cargo sorting, remain unclear. That said, recent work on a protein family of sorting nexins (SNX) – especially a subfamily of SNX that contain a BAR domain (SNX–BARs) – has begun to shed some much needed light on these issues and in particular the process of tubular–based endosomal sorting. SNX–BARs are evolutionary conserved in endosomal protein complexes such as retromer, where they co–ordinate membrane deformation with cargo selection. Furthermore a central theme emerges of SNX–BARs linking the forming membrane carrier to cytoskeletal elements for transport through motor proteins such as dynein. By studying these SNX–BARs, we are gaining an increasingly detailed appreciation of the mechanistic basis of endosomal sorting and how this highly dynamic process functions in health and disease.
- Published
- 2010
50. The retromer complex
- Author
-
Peter J. Cullen and Naomi Attar
- Subjects
Cancer Research ,Chemistry ,Vesicular Transport Proteins ,Lipid bilayer fusion ,Endosomes ,Computational biology ,Membrane Fusion ,Transport protein ,Fungal Proteins ,Wnt Proteins ,Retromer complex ,Protein Transport ,Multiprotein Complexes ,Genetics ,Animals ,Humans ,Molecular Medicine ,Disease ,Molecular Biology ,Plant Proteins ,Signal Transduction ,trans-Golgi Network - Published
- 2010
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.