SUMOylation proteomic analysis of renal distal convoluted tubule cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Epithelial Cell, Cell Culture
SUBMITTER: 
Qi Wu
LAB HEAD: Qi Wu
PROVIDER: PXD028507 | Pride | 2021-11-25
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 071016_2_Lys-C_HCD_OT_Lei_2h_50cm.raw | Raw | |||
| 071016_3_Glu-C_HCD_OT_Lei_2h_50cm.raw | Raw | |||
| DCT-S1-GluC-KGG-A.raw | Raw | |||
| DCT-S1-GluC-KGG-A_190211095135.raw | Raw | |||
| DCT-S1-GluC-KGG-B.raw | Raw |
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Project description:We profiled SUMO1 and SUMO2 modified proteins in cortical collecting duct (mpkCCD) as a precursor to targeted approaches to assess SUMO function. SUMO levels in cells following heat shock and treatment with deSUMOylation inhibitors were assessed.
2019-10-07 | PXD013346 | Pride
Project description:The distal convoluted tubule (DCT) is a short nephron segment, interposed between the macula densa and collecting duct. Even though it is short, it plays a key role in regulating extracellular fluid volume and electrolyte homeostasis. DCT cells are rich in mitochondria, and possess the highest density of Na+/K+-ATPase along the nephron, where it is expressed on the highly amplified basolateral membranes. DCT cells are largely water impermeable, and reabsorb sodium and chloride across the apical membrane via electroneurtral pathways. Prominent among this is the thiazide-sensitive sodium chloride cotransporter, target of widely used diuretic drugs. These cells also play a key role in magnesium reabsorption, which occurs predominantly, via a transient receptor potential channel (TRPM6). Human genetic diseases in which DCT function is perturbed have provided critical insights into the physiological role of the DCT, and how transport is regulated. These include Familial Hyperkalemic Hypertension, the salt-wasting diseases Gitelman syndrome and EAST syndrome, and hereditary hypomagnesemias. The DCT is also established as an important target for the hormones angiotensin II and aldosterone; it also appears to respond to sympathetic-nerve stimulation and changes in plasma potassium. Here, we discuss what is currently known about DCT physiology. Early studies that determined transport rates of ions by the DCT are described, as are the channels and transporters expressed along the DCT with the advent of molecular cloning. Regulation of expression and activity of these channels and transporters is also described; particular emphasis is placed on the contribution of genetic forms of DCT dysregulation to our understanding.
| S-EPMC5810970 | biostudies-literature
Project description:The kidney distal convoluted tubule (DCT) plays an important role in body sodium regulation and thus control of blood pressure. The main sodium reabsorption pathways in the DCT are the epithelial sodium channel (ENaC) and the thiazide-sensitive NaCl cotransporter (NCC), the functions of which can be modulated by the hormone vasopressin (VP) acting via uncharacterized signaling cascades. We performed large scale stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics of cultured mouse DCT cells (mpkDCT) to map global changes in protein phosphorylation events upon acute treatment with the VP type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP). The aim of this study is to identify unique VP signaling cascades in DCT cells that may be important for regulating ENaC or NCC activity. These pathways may be novel targets for modulation of blood pressure.
2015-08-04 | PXD001729 | Pride
Project description:The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane-associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long-term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD.
2017-12-15 | PXD005454 | Pride
Project description:Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2 and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 60,000 acceptor sites for ubiquitin and 40,000 acceptor sites for SUMO2/3. However, E3-to-target wiring is poorly understood. The limited number of SUMO E3s provides the unique opportunity to systematically study E3-substrate wiring. We developed SUMO Activated Target Traps (SATTs) and, in the presence of proteasome inhibitor MG132,systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL(ZNF451-3) and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference, even at the substrate isoform level. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool (https://amsterdamstudygroup.shinyapps.io/polarVolcaNoseR_revised/) to browse the dataset in an interactive manner, increasing the accessibility of this resource for the community. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.
2023-08-03 | PXD038326 | Pride
Project description:BackgroundThe distal convoluted tubule (DCT) plays an indispensable role in magnesium (Mg2+) reabsorption in the kidney. Yet, the extrusion mechanism of Mg2+ has not been identified. The solute carrier 41A3 (SLC41A3) has been suggested to be involved in Mg2+ extrusion, but this has never been conclusively demonstrated.MethodsUsing available RNA-sequencing data and real-time quantitative PCR, expression of two alternative Slc41a3 transcripts, encoding isoform (Iso) 1 or 2, were assessed in kidney and isolated DCT tubules. HEK293 or HAP1 cells were transfected with plasmids expressing either of the isoforms, followed by 25Mg2+ transport studies. Identification of cis-regulatory elements (CRE) was achieved by combining data from publicly available ATAC sequencing data and luciferase assays.ResultsGene expression studies revealed a distinct transcript of Slc41a3 in the DCT with an alternative promoter, leading to a protein with a unique N-terminus; SLC41A3-Iso 2. HEK293 cells overexpressing SLC41A3-Iso 2, but not -Iso 1, exhibited 2.7-fold and 1.6-fold higher 25Mg2+ uptake and extrusion, compared to mock, respectively. The transport was independent of Na+, of the Mg2+ channel TRPM7 or of transporters CNNM3 and -4. We identified a CRE accessible in the DCT, ±2.8kb upstream of the transcript. The presence of the CRE increased the Slc41a3-Iso 2 promoter activity 3.8-fold following luciferase assays, indicating the CRE contains an enhancer function.ConclusionIn conclusion, we identified two alternative transcripts of Slc41a3 in mouse. Slc41a3-Iso 2 is enriched within the DCT using specific gene regulatory elements. We speculate that specifically in the DCT, SLC41A3-Iso 2 orchestrates Mg2+ extrusion.
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Project description:BackgroundThe potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown.MethodsWe used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both.ResultsDeletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice.ConclusionsNedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.
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Project description:We review a collection of published renal epithelial transport models, from which we build a consistent and reusable mathematical model able to reproduce many observations and predictions from the literature. The flexible modular model we present here can be adapted to specific configurations of epithelial transport, and in this work we focus on transport in the proximal convoluted tubule of the renal nephron. Our mathematical model of the epithelial proximal convoluted tubule describes the cellular and subcellular mechanisms of the transporters, intracellular buffering, solute fluxes, and other processes. We provide free and open access to the Python implementation to ensure our multiscale proximal tubule model is accessible; enabling the reader to explore the model through setting their own simulations, reproducibility tests, and sensitivity analyses.
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Project description:Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR) but not the nucleoside analog Cytarabine, the two main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 hours) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, accelerating deSUMOylation dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that proteins that are deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. Although DNR does not modify CTCF binding on chromatin in general and at NFKB2 promoter in particular, it leads to a SUMO-dependent reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with distal cis-regulatory regions.
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Project description:SILAC was employed to investigate the effect of aldosterone stimulation on SUMOylation of cortical collecting duct (mpkCCD) cells.
2019-10-07 | PXD013363 | Pride