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A slit-diaphragm-associated protein network for dynamic control of renal filtration

ABSTRACT: The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm (SD), a proteinaceous junction between the foot processes of renal podocytes. Based on studies of structure and function the SD has been hypothesized to operate as a final barrier or as a ‘molecular sensor’ of renal filtration. Using high-resolution proteomic analysis of SDs affinity-isolated from rodent kidney, we show that the native SD is built from the known junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling receptors, kinases and phosphatases, transporters and scaffolds. Knock-out or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide receptor C (ANPRC) and protein ITM2B resulted in target-specific impairment or disruption of the filtration process. Thus, our results identify the SD as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Rattus Norvegicus (rat) Mus Musculus (mouse)

TISSUE(S): Kidney

SUBMITTER: Alexander Haupt

LAB HEAD: Prof. Dr. Bernd Fakler

PROVIDER: PXD034356 | Pride | 2022-09-20

REPOSITORIES: Pride

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Project description:Previous systems-based proteomic approaches have characterized alterations in protein co-expression networks of unfractionated asymptomatic (AsymAD) and symptomatic Alzheimer’s disease (AD) brains. However, it remains unclear how sample fractionation and sub-proteomic analysis influences the organization of these protein networks and their relationship to clinicopathological traits of disease. In this proof-of-concept study, we performed a systems-based sub-proteomic analysis of membrane-enriched post-mortem brain samples from pathology-free control, AsymAD, and AD brains (n=6 per group). Label-free mass spectrometry based on peptide ion intensity was used to quantify the 18 membrane-enriched fractions. Differential expression and weighted protein co-expression network analysis (WPCNA) were then used to identify and characterize modules of co-expressed proteins most significantly altered between the groups. We identified a total of 27 modules of co-expressed membrane-associated proteins. In contrast to the unfractionated proteome, these networks did not map strongly to cell-type specific markers. Instead, these modules were principally organized by their associations with a wide variety of membrane-bound compartments and organelles. Of these, the mitochondrion was associated with the greatest number of modules, followed by modules linked to the cell surface compartment. In addition, we resolved networks with strong associations to the endoplasmic reticulum, Golgi apparatus, nucleus, and other membrane-bound organelles. Fourteen of the 27 modules demonstrated significant correlations with clinical and pathological AD phenotypes. These results revealed that the proteins within individual compartments feature a heterogeneous array of AD-associated expression patterns, particularly during preclinical stages of disease. In conclusion, this systems-based analysis of the membrane-associated AsymAD brain proteome yielded a unique network organization highly linked to cellular compartmentalization. Further study of this membrane-associated proteome may reveal novel insight into the complex pathways governing the earliest stages of disease.

Project description:While modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors; thus, reducing overall radiation exposure to normal tissues, moderate dose and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in Human Coronary Artery Endothelial Cells (HCAECs) using single-dose irradiation (SD, 10Gy) or multi-fractionated irradiation (MF, 2Gy x 5) regimens. Longitudinal timepoints were collected after a SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared to SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis (IPA) to uncover miRs associated with target transcripts from several cellular pathways post-irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. Additionally, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events and angiogenesis were revealed. Human Coronary Artery Endothelial Cells (HCAECs) were irradiated in a PANTAK high frequency X-ray generator (Precision X-ray Inc., N. Bedford, CT), operated at 300kV and 10MA. The dose rate was 1.6 Gy per min. Cells were plated into T75cm2 flasks (1-1.5 x 10^6 for single dose radiation and 0.6-0.8 x 10^6 for fractionated radiation). After 24h, cells were exposed to a total of 10 Gy radiation administered either as a single-dose radiation (SD), or as multi-fractionated radiation of 2 Gy x 5 (MF). These non-isoeffective doses were selected to simulate clinical hypofractionated and conventionally fractionated radiotherapy regimens. For the MF protocol, cells were exposed to 2 Gy radiation twice a day, at 6h interval. The cells were approximately 90% confluent at the time of harvesting. For both protocols, radiation-induced changes were analyzed at 6h and 24h after a SD and 6h and 24h after the final dose of fractionated irradiation. Separate controls were maintained for SD and MF radiation protocols.

			Action	DRS
	F131822.dat	Other
	F131822.mzId.gz	Mzid
	F131824.dat	Other
	F131824.mzId.gz	Mzid
	F131825.dat	Other

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A slit-diaphragm-associated protein network for dynamic control of renal filtration

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