Dataset Information

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: David Stroud

LAB HEAD: David Stroud

PROVIDER: PXD036424 | Pride | 2022-11-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	191217_Joanna_JS-288-1.raw	Raw
	191217_Joanna_JS-288-2.raw	Raw
	191217_Joanna_JS-288-3.raw	Raw
	191217_Joanna_JS-288-4.raw	Raw
	191217_Joanna_JS-288-5.raw	Raw

Items per page:

1 - 5 of 8

Publications

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins.

Healy Michael D MD Sacharz Joanna J McNally Kerrie E KE McConville Calum C Tillu Vikas A VA Hall Ryan J RJ Chilton Molly M Cullen Peter J PJ Mobli Mehdi M Ghai Rajesh R Stroud David A DA Collins Brett M BM

Structure (London, England : 1993) 20221026 12

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 ...[more]

PMID: 36302387

Similar Datasets

Project description:PDZ (Post-synaptic density, PSD-95)/ Disc large, Dlg/ Zonula occludens, ZO-1) proteins are central in the assembly of multiprotein signalling complexes that are formed in distinctive, specialized cell regions. In these complexes different signals are orchestrated and traduced into quick and efficient responses controlling cell polarization and cell communication. Viral pathogens target host PDZ proteins by expressing viral proteins containing a PDZ binding motif (PBM). SARS-CoV-1 and -2 harbour the protein E, a viroporin with a conserved PBM in its carboxyl-terminal region. SARS-CoV-1 E protein is a pathogenicity factor that in epithelia interacts with the PDZ protein PALS1, which promote disruption of cell junctions. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes, dendritic cells, and alveolar macrophages. Identification of targets of SARS-CoV-2 E protein in immune cells might offer valuable clues to understand how SARS-CoV-2 alters immune response. Here we generated a SARS-CoV-2 E protein fused to a GFP-tag at the amino terminal. This recombinant protein was used as bait to identify associated proteins in THP-1 cells, a human monocytic cell line that can be differentiated to macrophages and dendritic cells. Analysis of the GFP-E protein interactome provided 372 proteins that fall into different functional groups. Only eight of these proteins harbor PDZ domains, including the cell polarity protein ZO-2 and the chemoattractant IL-16. Syntenin, a PDZ protein previously identified as interactor of SARS-CoV-1 E protein in epithelia, was also found in our analysis. Little is known about these PDZ proteins in immune cells, so we addressed their expression in monocytes and along the differentiation towards macrophages and dendritic cells. Our findings support the notion that viral targeting of PDZ proteins alters inflammatory response and highlight the importance of identifying the functions of PDZ proteins into maintenance of immune fitness.

Project description:We performed chromatin immunoprecipitation on microarray (ChIP-chip) transcriptional profiling of the SsrB regulator of Salmonella enterica Typhimurium to better characterize its regulon and to identify the DNA-recognition element coordinating its specific interaction at cis-regulatory sites. SsrB, the response regulator of the two component regulatory system SsrA-SsrB encoded within the Salmonella Pathogenicity Island (SPI-2), directs transcriptional activation of the closely associated type three secretion system (T3SS) also encoded at this locus. Immunoprecipitaiton of SsrB translationally fused to a C-terminal FLAG-tag from formaldehyde cross-linked genomic DNA was performed under SPI-2 activating and non-activating conditions. Downstream analysis and experimental work identified specific interactions within SPI-2 at the previously identified ssrA, ssaB, sseA, ssaG, ssaM promoters and identified an additional cryptic promoter driving expression upstream of ssaR. Additionally, a previously unknown SsrB interaction site within ssaE was shown to be the major contributor driving the expression of the downstream genes and not the previously identified interaction site within the intergenic region of ssaE-sseA. Interactions were also identified outside of SPI-2 upstream of other T3SS-associated genes encoded within other genomic islands, and for other uncharacterized genes. Integration of this data with transcriptional microarray work, previously published DNase I footprinting data, bacteria 1-hybrid investigations and comparative genomics analyses of cis-regulatory regions within the orthologous Sodalis symbiosis region 3 (SSR-3) of Sodalis glossinidius enabled identification of a conserved 18bp palindrome which was experimentally validated as being required for transcriptional activation of SsrB dependant genes. SsrB-FLAG immunoprecipitations were performed under SsrB activating and non-activating conditions from formaldehyde cross-linked bacterial lysates. Nine immunoprecipitation reactions were pooled into three replicate samples for the activating condition and three reactions were pooled into one sample for the non-activating control condition. The immunoprecipitated DNA in addition to the non-immunoprecipitated control DNA for each of the four samples were labeled with Cy3 or Cy5 fluorophores respectively and were concurrently hybridized to four arrays on a single slide.