Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1ϩ vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

We initially selected 35 cases (pilot cohort) of PLA2R-negative MN on kidney biopsy for analysis by tandem mass spectrometry (MS/MS), and detected the unique protein, NELL-1,

The progesterone receptor can be reconstituted into hsp90-containing complexes in vitro, and the resulting complexes are needed to maintain hormone binding activity. This process requires ATP/Mg 2؉ , K ؉ , and several axillary proteins. We have developed a defined system for the assembly of progesterone receptor complexes using purified proteins. Five proteins are needed to form complexes that are capable of maintaining hormone binding activity. These include hsp70 and its cochaperone, hsp40, the hsp70/hsp90-binding protein, Hop, hsp90, and the hsp90-binding protein, p23. The proteins Hip and FKBP52 were not required for this in vitro process even though they have been observed in receptor complexes. Each of the five proteins showed a characteristic concentration dependence. Similar concentrations of hsp70, hsp90, and p23 were needed for optimal assembly, but hsp40 and Hop were effective at about 1/10 the concentration of the other proteins, suggesting that these two proteins act catalytically or are needed at levels similar to the receptor concentration. ATP was required for the functioning of both hsp70 and hsp90. The binding of hsp70 to the receptor requires hsp40 and about 10 M ATP; however, hsp90 binding appears to occur subsequent to hsp70 binding and is optimal with 1 mM ATP. A three-step model is presented to describe the assembly process.When extracted from tissue cytosol, receptors for progesterone (PR), 1 glucocorticoid (GR), and other steroids exist in heteromeric complexes containing heat shock protein 90 (hsp90) and several additional proteins (1-3). Recent information on the assembly of these complexes has been gained mainly through the use of an in vitro system consisting of immuneisolated progesterone (4) or glucocorticoid (5) receptor incubated in rabbit reticulocyte lysate. The formation of receptor complexes in this system is dependent upon ATP hydrolysis, the ions Mg 2ϩ and K ϩ , and the participation of a number of proteins (6, 7). When assembled in vitro, the mature avian progesterone receptor complex closely resembles that obtained from oviduct cytosol and contains hsp90, any one of three immunophilins, and a 23-kDa phosphoprotein, p23. It also contains variable sub-stoichiometric amounts of hsp70. The immunophilins include the cyclosporin A-binding protein, CyP-40, and two FK506-binding proteins, FKBP51 and FKBP52. Antibody inhibitor studies or depletion and reconstitution experiments indicate that hsp70 (6, 8), Hop, an intermediate in complex assembly (9), and p23 (10, 11) are essential for the formation of hsp90 complexes, but the actual roles of these and other proteins are still unclear.An intermediate in PR complex formation has been identified that contains submaximal amounts of hsp90, substantially more hsp70 than in the mature complex, and two additional proteins, Hop (p60) and Hip (p48) (6, 12). This complex does not contain immunophilins or p23. Hop (hsp-organizing protein) is a 60-kDa stress-related protein that binds to both hsp70 and hsp90 (9, 13-15). Hip (hsp70-interacting ...