Dataset Information

Regulation of the endosomal SNX27 retromer by OTULIN

ABSTRACT: OTULIN specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Using mass spectrometry, we discovered an interaction of OTULIN with SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the retromer subunit VPS26 and endosome-to-membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of retromer assembly and protein recycling to the cell surface.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, T Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Adan Pinto-Fernandez

LAB HEAD: Benedikt Kessler

PROVIDER: PXD012082 | Pride | 2019-09-25

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	HF247_MSQ1052_AureliaWeber_S5_1.raw	Raw
	HF247_MSQ1052_AureliaWeber_S5_2.raw	Raw
	HF247_MSQ1052_AureliaWeber_S5_3.raw	Raw
	HF247_MSQ1052_AureliaWeber_S5_4.raw	Raw
	HF247_MSQ1052_AureliaWeber_S6_1.raw	Raw

Items per page:

1 - 5 of 22

Publications

Regulation of the endosomal SNX27-retromer by OTULIN.

Stangl Aurelia A Elliott Paul R PR Pinto-Fernandez Adan A Bonham Sarah S Harrison Luke L Schaub Annalisa A Kutzner Kerstin K Keusekotten Kirstin K Pfluger Paul T PT El Oualid Farid F Kessler Benedikt M BM Komander David D Krappmann Daniel D

Nature communications 20190920 1

OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ do ...[more]

PMID: 31541095

Similar Datasets

Project description:T lymphocyte recognition of antigenic peptides on the surface of antigen-presenting cells (APC) leads to immune synapse formation (IS). By analogy with the nervous synapse, this cell-cell communication model requires the formation of highly organized structures that delimit an active zone of membrane traffic. For T cells to connect antigen recognition with the biological response, receptors and signaling proteins must be recycled. Sorting nexin 27 (SNX27) is an endosomal protein best known for its role in recycling PDZ (PSD95, Dlg1, ZO-1)-interacting neuronal proteins via the retromer transport machinery. Alteration of this sorting pathway leads to neurodegeneration and is associated to Down syndrome, Alzheimer’s and Parkinson’s diseases. In T lymphocytes, SNX27 interacts with diacylglycerol kinase zeta (DGKz), which provides a mechanistic link between diacylglycerol metabolism and membrane trafficking. Following antigen recognition, SNX27-positive endosomes polarize to the IS and a PDZ-dependent SNX27 pool accumulates at the cell-cell contact area. To test for additional SNX27 functions, we carried out proteomic analysis of the SNX27 interactome purified from T cells in contact with APC, and identified SNX27 interaction with the retromer and with members of the WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) complex. This finding indicates the conserved nature of the SNX27/WASH/retromer complex in hematopoietic cells and suggests similarity between neurological diseases and abnormal immune/inflammatory responses. We also found PDZ-dependent SNX27 cargoes, including modulators of cytoskeletal reorganization such as zona occludens-2 (ZO-2). ZO-2, a cytosolic component of epithelial cell-cell junctions, localized to the IS, and SNX27 silencing affected ZO-2 stability at the synapse. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in epithelial systems also participate in IS regulation.

Project description:Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human glucocorticoid receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t1/2=8±1 min vs. 62±17 min in DMSO-treated cells) and CCL7 mRNA (t1/2=15±4 min vs. 114±37 min), but not that of CCL5 mRNA (t1/2=231±8 min vs. 266±5 min) in the BEAS-2B cell line. This effect was inhibited by pre-incubation with an anti-GR antibody, indicating that GR itself plays a role in the turnover of these transcripts. Co-immunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5’UTR of CCL2 mRNA, and further mapped to nucleotides 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein. The BEAS-2B cell line is derived from human tracheal epithelium transformed by an adenovirus 12-SV40 hybrid virus. For isolation of glucocorticoid receptor-associated transcripts, cytoplasmic lysates obtained from 1.6x107 cells/condition, 4 replicates per condition, were used to generate a collection of transcripts associated with GR by immunoprecipitation of the GR-mRNA complexes. The RNA from these IP's were extracted and labeled with the Illumina TotalPrep RNA Amplification Kit which generates biotin-labeled cRNA. This was hybridized to Illumina's Sentrix HumanRef-8,v3 Expression BeadChips. The data from 3 replicates of GR-IP and 4 replicates of Control-IP were used for global analysis of GR-mRNA interactions.