From Molecules to Mechanisms: Functional Proteomics and Its Application to Renal Tubule Physiology.

Classical physiological studies using electrophysiological, biophysical, biochemical, and molecular techniques have created a detailed picture of molecular transport, bioenergetics, contractility and movement, and growth, as well as the regulation of these processes by external stimuli in cells and organisms. Newer systems biology approaches are beginning to provide deeper and broader understanding of these complex biological processes and their dynamic responses to a variety of environmental cues. In the past decade, advances in mass spectrometry-based proteomic technologies have provided invaluable tools to further elucidate these complex cellular processes, thereby confirming, complementing, and advancing common views of physiology. As one notable example, the application of proteomics to study the regulation of kidney function has yielded novel insights into the chemical and physical processes that tightly control body fluids, electrolytes, and metabolites to provide optimal microenvironments for various cellular and organ functions. Here, we systematically review, summarize, and discuss the most significant key findings from functional proteomic studies in renal epithelial physiology. We also identify further improvements in technological and bioinformatics methods that will be essential to advance precision medicine in nephrology.

AbDesigner3D: a structure-guided tool for peptide-based antibody production.

Summary: We present AbDesigner3D, a new tool for identification of optimal immunizing peptides for antibody production using a peptide-based strategy. AbDesigner3D integrates 3D structural data from the Protein Data Bank (PDB) with UniProt data, which includes basic sequence data, post-translational modification sites, SNP occurrences and more. Other features, such as uniqueness and conservation scores, are calculated based on sequences from UniProt. The 3D visualization capabilities allow an intuitive interface, while an abundance of quantitative output simplifies the process of comparing immunogen peptides. Important quantitative features added in this tool include calculation and display of accessible surface area (ASA) and protein-protein interacting residues (PPIR). The specialized data visualization features of AbDesigner3D will greatly assist users to optimize their choice of immunizing peptides. Availability and implementation: AbDesigner3D is freely available at http://sysbio.chula.ac.th/AbDesigner3D or https://hpcwebapps.cit.nih.gov/AbDesigner3D/. Supplementary information: Supplementary data are available at Bioinformatics online.

Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus.

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

A machine learning strategy for predicting localization of post-translational modification sites in protein-protein interacting regions.

BACKGROUND: One very important functional domain of proteins is the protein-protein interacting region (PPIR), which forms the binding interface between interacting polypeptide chains. Post-translational modifications (PTMs) that occur in the PPIR can either interfere with or facilitate the interaction between proteins. The ability to predict whether sites of protein modifications are inside or outside of PPIRs would be useful in further elucidating the regulatory mechanisms by which modifications of specific proteins regulate their cellular functions. RESULTS: Using two of the comprehensive databases for protein-protein interaction and protein modification site data (PDB and PhosphoSitePlus, respectively), we created new databases that map PTMs to their locations inside or outside of PPIRs. The mapped PTMs represented only 5 % of all known PTMs. Thus, in order to predict localization within or outside of PPIRs for the vast majority of PTMs, a machine learning strategy was used to generate predictive models from these mapped databases. For the three mapped PTM databases which had sufficient numbers of modification sites for generating models (acetylation, phosphorylation, and ubiquitylation), the resulting models yielded high overall predictive performance as judged by a combined performance score (CPS). Among the multiple properties of amino acids that were used in the classification tasks, hydrophobicity was found to contribute substantially to the performance of the final predictive models. Compared to the other classifiers we also evaluated, the SVM provided the best performance overall. CONCLUSIONS: These models are the first to predict whether PTMs are located inside or outside of PPIRs, as demonstrated by their high predictive performance. The models and data presented here should be useful in prioritizing both known and newly identified PTMs for further studies to determine the functional relationship between specific PTMs and protein-protein interactions. The implemented R package is available online ( http://sysbio.chula.ac.th/PtmPPIR ).

Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-ß receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

Regulatory landscape of AGE-RAGE-oxidative stress axis and its modulation by PPARγ activation in high fructose diet-induced metabolic syndrome.

BACKGROUND: The AGE-RAGE-oxidative stress (AROS) axis is involved in the onset and progression of metabolic syndrome induced by a high-fructose diet (HFD). PPARγ activation is known to modulate metabolic syndrome; however a systems-level investigation looking at the protective effects of PPARγ activation as related to the AROS axis has not been performed. The aim of this work is to simultaneously characterize multiple molecular parameters within the AROS axis, using samples taken from different body fluids and tissues of a rat model of HFD-induced metabolic syndrome, in the presence or absence of a PPARγ agonist, Rosiglitazone (RGZ). METHODS: Rats were fed with 60% HFD for the first half of the treatment duration (21 days) then continued with either HFD alone or HFD plus RGZ for the second half. RESULTS: Rats receiving HFD alone showed metabolic syndrome manifestations including hypertension, dyslipidemia, increased glucose levels and insulin resistance, as well as abnormal kidney and inflammatory parameters. Systolic blood pressure, plasma triglyceride and glucose levels, plasma creatinine, and albuminuria were significantly improved in the presence of RGZ. The following molecular parameters of the AROS axis were significantly upregulated in our rat model: carboxymethyl lysine (CML) in urine and liver; carboxyethyl lysine (CEL) in urine; advanced glycation end products (AGEs) in plasma; receptor for advanced glycation end products (RAGE) in liver and kidney; advanced oxidation protein products (AOPP) in plasma; and 4-hydroxynonenal (HNE) in plasma, liver, and kidney. Conversely, with RGZ administration, the upregulation of AOPP and AGEs in plasma, CML and CEL in urine, RAGE in liver as well as HNE in plasma and liver was significantly counteracted/prevented. CONCLUSIONS: Our data demonstrate (i) the systems-level regulatory landscape of HFD-induced metabolic syndrome involving multiple molecular parameters, including HNE, AGEs and their receptor RAGE, and (ii) attenuation of metabolic syndrome by PPARγ modulation.

p120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).

Protein ubiquitinylation plays a key role in many important cellular processes. Ubiquitinylation requires the E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and, frequently, a substrate-specific E3 ubiquitin-protein ligase. In one class of E3 ubiquitin ligases, the catalytic domain contains a zinc-binding RING finger motif. ARD1 (ADP-ribosylation factor domain protein 1), with a RING finger domain in the N-terminal region, two predicted B-Boxes, and a coiled-coil protein interaction motif immediately preceding an ADP-ribosylation factor domain at the C terminus, belongs to the TRIM (Tripartite motif) or RBCC (RING, B-Box, coiled-coil) family. The region containing the B-Boxes and the coiled-coil motif acts as a GTPase-activating protein for the ADP-ribosylation factor domain of ARD1. We report here that full-length ARD1 or the RING finger domain (residues 1-110) produced polyubiquitinylated proteins in vitro in the presence of mammalian E1, an E2 enzyme (UbcH6 or UbcH5a, -5b, or -5c), ATP, and ubiquitin. Deletion of the RING region or point mutations within the RING sequence abolished ARD1 E3 ligase activity. All data are consistent with a potential function for ARD1 as an E3 ubiquitin ligase in cells.

Differential protein expression in lens epithelial whole-mounts and lens epithelial cell cultures.

PURPOSE: Lens fibergenesis is a problem in several types of cataract and in the posterior capsular opacification following cataract surgery. To correct improper fiber differentiation or to prevent unwanted growth on the posterior capsule following cataract surgery requires a thorough understanding of normal and abnormal fiber formation. To this end, studies were initiated to characterize fiber differentiation in the bovine lens and in lens epithelial cell cultures. METHODS: Indirect immunofluorescence and immunoblot analysis were employed to study the expression of vimentin, beta-crystallin, gamma-crystallin, filensin, aquaporin 0 and the Na, K-ATPase catalytic subunit isoforms (alpha1, alpha2, alpha3) in bovine lens epithelium whole-mounts as well as lens epithelial cell cultures propagated in medium containing 10% bovine serum or in medium supplemented with bovine serum concentrations < or =4%. RESULTS: Three distinct cell types were observed in the bovine lens epithelium. The cells of the central zone were identified by a polarized distribution of two distinct Na, K-ATPase catalytic subunit isoforms, alpha1 to the apical (fiber side) and alpha3 to the basal (aqueous humor side) membranes. Lateral to the polarized central zone, was the germinative zone of cells, best characterized by perinuclear vimentin basket-like structures and the loss of polarized Na, K-ATPase catalytic subunit isoforms. Lateral to the germinative zone were the cells of the transition zone (meridinal rows) where expression of the lens specific proteins beta-crystallin, gamma-crystallin, filensin and aquaporin 0 as well as the lens fiber-, adipocyte- and brain glia-specific Na, K-ATPase catalytic subunit, alpha2 are expressed. The cultured cells propagated in medium supplemented with 10% serum bore no resemblance to any of the cells of the bovine lens epithelium whole-mounts. The cells propagated in the medium supplemented with the lower bovine serum levels resembled the differentiating fibers of the transition zone of the bovine lens epithelium whole-mounts as well as superficial cortical fibers. CONCLUSIONS: Since the low-serum lens epithelial cell cultures bear a remarkable resemblance to early differentiating fibers, they are reasonable models for the study of early fiber differentiation or prevention of differentiation. The culture conditions employed do not yield the polarized cells of the central zone. Nor has the function of these polarized cells in lens fluid, nutrient and ion homeostasis been determined.