Large-scale identification of calcium oxalate monohydrate crystal-binding proteins on apical membrane of distal renal tubular epithelial cells

Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na(+)/K(+)-ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets.     

Calcium oxalate dihydrate crystal induced changes in glycoproteome of distal renal tubular epithelial cells

Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.     

Altered proteins in MDCK renal tubular cells in response to calcium oxalate dihydrate crystal adhesion: a proteomics approach

The interaction between crystals and renal tubular cells has been proposed to be a crucial event that elicits subsequent cellular responses, leading to kidney stone formation. Nevertheless, the molecular mechanisms of these cellular responses remain poorly understood. We performed a gel-based differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby canine kidney (MDCK) cells, which were derived from dog kidney and exhibited distal renal tubule phenotype, during calcium oxalate dihydrate (COD) crystal adhesion. MDCK cells were grown in a medium without or with COD crystals (100 microg/ml) for 48 h. Crystal adhesion was illustrated by phase-contrast and scanning electron microscopy. Flow cytometry using annexin V/propidium iodide double staining showed that the percentage of cell death did not significantly differ between cells with and without COD crystal adhesion. Cellular proteins were then extracted, resolved with two-dimensional gel electrophoresis (2-DE), and visualized by SYPRO Ruby staining ( n = 5 gels per group). Quantitative intensity analysis revealed 11 significantly altered proteins, 10 of which were successfully identified by quadrupole time-of-flight peptide mass fingerprinting (MS) and/or tandem MS (MS/MS), including metabolic enzymes, cellular structural protein, calcium-binding protein, adhesion molecule, protein involved in RNA metabolism, and chaperone. An increase in annexin II was confirmed by 2-D Western blot analysis. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COD crystal adhesion.     

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.     

Proteomic analysis of calcium oxalate monohydrate crystal-induced cytotoxicity in distal renal tubular cells

Calcium oxalate monohydrate (COM) is the major crystalline component found in kidney stones and its adhesion to renal tubular cells provokes tubular injury, which in turn enhances COM crystal adhesion. However, COM-induced toxic effects in these tubular cells remain largely unknown. We performed a proteomics study to characterize changes in the cellular proteome in MDCK distal renal tubular cells after an exposure to high-dose (1000 microg/mL) COM crystals for 48 h, at which percentage of cell death was significantly increased. Proteins were extracted from MDCK cells cultured with COM-containing or COM-free medium ( n = 5 individual flasks per group), resolved in individual 2-D gels, and stained with SYPRO Ruby fluorescence dye. Quantitative and statistical analyses revealed 53 proteins whose abundance levels were altered (25 were increased, whereas other 28 were decreased) by COM-induced toxicity. Among these, 50 were successfully identified by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and/or tandem MS (MS/MS) analyses. The proteomic data were clearly confirmed by 2-D Western blot analysis. While three chaperones (GRP78, Orp150 and Hsp60) were increased, other proteins involved in protein biosynthesis, ATP synthesis, cell cycle regulator, cellular structure, and signal transduction were decreased. These data provide some novel mechanistic insights into the molecular mechanisms of COM crystal-induced tubular toxicity.     

Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells

Dengue virus requires the presence of an unidentified cellular receptor on the surface of the host cell. By using a recently published affinity chromatography approach, an 84-kDa molecule, identified as heat shock protein 90 (HSP90) by matrix-assisted laser desorption ionization-time of flight mass spectrometry, was isolated from neuroblastoma and U937 cells. Based on the ability of HSP90 (84 kDa) to interact with HSP70 (74 kDa) on the surface of monocytes during lipopolysaccharide (LPS) signaling and evidence that LPS inhibits dengue virus infection, the presence of HSP70 was demonstrated in affinity chromatography eluates and by pull-down experiments. Infection inhibition assays support the conclusion that HSP90 and HSP70 participate in dengue virus entry as a receptor complex in human cell lines as well as in monocytes/macrophages. Additionally, our results indicate that both HSPs are associated with membrane microdomains (lipid rafts) in response to dengue virus infection. Moreover, methyl-beta-cyclodextrin, a raft-disrupting drug, inhibits dengue virus infection, supporting the idea that cholesterol-rich membrane fractions are important in dengue virus entry.     

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.     

High calcium enhances calcium oxalate crystal binding capacity of renal tubular cells via increased surface annexin A1 but impairs their proliferation and healing

Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further targeted functional studies to confirm their relevance in hypercalciuria and kidney stone disease in vivo.     

Reconstitution of the 9 S estrogen receptor with heat shock protein 90

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.     

Association of the Ah receptor with the 90-kDa heat shock protein

Partially purified Ah receptor preparations were used to produce a monoclonal antibody, designated as 8D3, that is capable of immunoprecipitating the Ah receptor. Hepa 1c1c7 cytosol was photoaffinity-labeled with [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin followed by immunoprecipitation, and the resulting precipitate was applied to a sodium dodecyl sulfate-polyacrylamide electrophoretic gel. These gels were stained with Coomassie Blue and revealed the presence of a major immunoprecipitated 90-kDa protein, and after autoradiography a radiolabeled 95-kDa protein (Ah receptor) was detected. The 90-kDa protein was determined to be the 90-kDa heat shock protein (HSP90) by western blot analysis using an antibody (AC88) previously shown to be specific for HSP90. An increase in the sedimentation of the Ah receptor on sucrose density gradients was seen upon addition of monoclonal antibody 8D3 to Hepa 1c1c7 cytosol. Monoclonal antibody 8D3 immunoprecipitates the Ah receptor from Hepa 1 cells (murine), HeLa cells (human), and rat liver cytosolic extracts, indicating that the Ah receptor is complexed with HSP90 in several mammalian species tested. These results illustrate another physicochemical property that the supergene family of soluble steroid receptors and the Ah receptor have in common.     

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.     

Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. 45Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.     

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [-³²P]ATP/Mg²⁺ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [-³²P]ATP·Mg²⁺ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 , and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 .     

Regulation of the atrial natriuretic peptide receptor by heat shock protein 90 complexes

Heat shock protein 90 (hsp90) is a chaperone required for the proper folding and trafficking of many proteins involved in signal transduction. We tested whether hsp90 plays a role as a chaperone for GC-A, the membrane guanylate cyclase that acts as a receptor for atrial natriuretic peptide (ANP). When cultured cells expressing recombinant GC-A were treated with geldanamycin, an inhibitor of hsp90 function, the ANP-stimulated production of cyclic GMP was inhibited. This suggested that hsp90 was required for GC-A processing and/or stability. A physical association between hsp90 and GC-A was demonstrated in coimmunoprecipitation experiments. Treatment with geldanamycin disrupted this association and led to the accumulation of complexes containing GC-A and heat shock protein 70 (hsp70). Protein folding pathways involving hsp70 and hsp90 include several pathway-specific co-chaperones. Complexes between GC-A and hsp90 contained the co-chaperone p50(cdc37), typically found associated with protein kinase.hsp90 heterocomplexes. GC-A immunoprecipitates did not contain detectable amounts of Hop, FKBP51, FKBP52, PP5, or p23, all co-chaperones found in hsp90 complexes with other signaling proteins. The association of hsp90 and p50(cdc37) with GC-A was dependent on the kinase homology domain of this receptor but not on its ANP-binding, transmembrane, or guanylate cyclase domains. The data suggest that GC-A is regulated by hsp90 complexes similar to those involved in the maturation of protein kinases.     

Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose

Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.