Dataset Information

Proteomic profiling in mouse kidneys post-UUO

ABSTRACT: Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of chronic kidney disease, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction, using TG2-knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome post-UUO detected by SWATH-mass spectrometry.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Kidney Cell, Kidney

DISEASE(S): Chronic Kidney Disease

SUBMITTER: David Boocock

LAB HEAD: David Boocock

PROVIDER: PXD008173 | Pride | 2018-02-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Kidney_IDA_KOf_Sham_Pool.wiff	Wiff
	Kidney_IDA_KOf_Sham_Pool.wiff.scan	Wiff
	Kidney_IDA_KOf_UUO_Pool.wiff	Wiff
	Kidney_IDA_KOf_UUO_Pool.wiff.scan	Wiff
	Kidney_IDA_WTf_Sham_Pool.scan	Other

Items per page:

1 - 5 of 256

Publications

Proteomic Profiling Reveals the Transglutaminase-2 Externalization Pathway in Kidneys after Unilateral Ureteric Obstruction.

Furini Giulia G Schroeder Nina N Huang Linghong L Boocock David D Scarpellini Alessandra A Coveney Clare C Tonoli Elisa E Ramaswamy Raghavendran R Ball Graham G Verderio Claudia C Johnson Timothy S TS Verderio Elisabetta A M EAM

Journal of the American Society of Nephrology : JASN 20180130 3

Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding p ...[more]

PMID: 29382685

Similar Datasets

Project description:Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in the pathogenesis of CD, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we developed a reciprocal proteomic analysis strategy, which facilitates the comprehensive identification of isopeptides in a complex protein hydrolysate. The workflow consists of four steps: (1) performance of the model reaction between TG2 and gluten peptide(s) followed by tryptic hydrolysis, clean-up and untargeted nLC-MS/MS analysis (2), prediction of tryptic TG2-derived lysine peptides that serve as potential isopeptide modifications (3) the search for isopeptides by configuring the TG2-modifications in MaxQuant and search against the α-gliadin fasta file and (4) the reciprocal search for isopeptides by configuring the gluten peptide(s) as modification in MaxQuant and search against the TG2 fasta file. After verification by manual data curation and visualization, this strategy enabled the identification of 26 different isopeptides involving 19 TG2 lysine residues, only six of which were known previously. Experiments with different TG2-gluten peptide molar ratios revealed that K-425, K-590, K-600 and K-649 were the most preferred lysine residues involved in isopeptide crosslinking. Further, expanding the model system to three gluten peptides allowed the localization of the preferred glutamine crosslinking sites. The described strategy is generally applicable to any hydrolysate containing isopeptides and these new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.

Project description:Rodents are commonly housed below thermoneutrality (~20°C) 1. Under these conditions there is a substantial effect on rodent physiology including the hyperactivation of brown (BAT) and beige adipose tissue 2. Here, we raised animals from weaning, on an obesogenic diet at thermoneutrality (28°C) to closer mimic human physiology and determine the impact of a) moderate cold exposure (i.e. 20°C, a temperature reduction of ~8°C) or b) treatment with YM-178, a highly-selective, clinically used β3-adrenoreceptor agonist on classical BAT or subcutaneous inguinal (IWAT) beige depots. Under these conditions, uncoupling protein 1 mRNA was undetectable in IWAT in all groups. Maintenance at 20°C drove weight gain and a 125% increase in subcutaneous fat, an effect not seen with YM-178 administration thus suggesting a direct effect of ambient temperature in promoting weight gain and adiposity in obese rats. Using exploratory adipose tissue proteomics we reveal novel processes and pathways associated with cold-induced weight gain in BAT (i.e. histone deacetylation and glycosphingolipid biosynthesis) and IWAT (i.e. NAD+ binding and retinol metabolism). Conversely, YM-178 had minimal metabolic-related effects on BAT and drove a pro-inflammatory phenotype in IWAT. Exercise training elicits diverse effects on brown (BAT) and white adipose tissue (WAT) physiology in rodents housed below their thermoneutral zone (i.e. 28-32°C). In these conditions, BAT is chronically hyperactive and, unlike human residence, closer to thermoneutrality. Therefore, we set out to determine the effects of exercise training in obese animals at 28°C (i.e. thermoneutrality) on BAT and WAT in its basal (i.e. inactive) state. Sprague-Dawley rats (n=12) were housed at thermoneutrality from 3 weeks of age and fed a high-fat diet. At 12 weeks of age half these animals were randomised to 4-weeks of swim-training (1 hour/day, 5 days per week). Following a metabolic assessment interscapular and perivascular BAT and inguinal (I)WAT were taken for analysis of thermogenic genes and the proteome. Exercise attenuated weight gain but did not affect total fat mass or thermogenic gene expression. Proteomics revealed an impact of exercise training on2-oxoglutarate metabolic process, mitochondrial respiratory chain complex IV, carbon metabolism and oxidative phosphorylation. This was accompanied by an upregulation of multiple proteins involved in skeletal muscle physiology suggesting an adipocyte to myocyte switch in BAT. UCP1 mRNA was undetectable in IWAT with proteomics highlighting changes to DNA binding, the positive regulation of apoptosis, HIF-1 signalling and cytokine-cytokine receptor interaction.