Clearance of p-cresol sulfate and β-2-microglobulin from dialysate by commercially available sorbent technology

Dialysate regeneration by sorbents is an alternative to conventional single-pass dialysis. Little is known about the capacity of sorbents to clear dialysate of “middle molecules” and protein-bound uremic toxins. We studied p-cresol sulfate (PCS) and β-2-microglobulin (β2M) removal from dialysate by a sorbent: 1. PCS (40 mg PCS dissolved in 4 L of fresh dialysate) was recirculated through a sorbent cartridge (SORB Technology, Inc.) for analysis of PCS removal. 2. Spent peritoneal dialysate was recirculated on the “blood” side of a high-flux dialyzer. On the “dialysate” side of the membrane, bicarbonate dialysate was recirculated through a sorbent cartridge. β2M was measured in both streams. Two results are of particular importance for the use of regenerated fluid in chronic dialysis: 1. PCS was virtually completely removed from the dialysate. On average, PCS concentration was reduced to 1.4% of the starting concentration after 60 minutes. PCS extraction across the sorbent was nearly complete at any time. 2. β2M was on average reduced to 14.3% of the starting concentration after 60 minutes. Postsorbent concentrations were consistently below the validated range of the test method. We conclude that PCS and β2M are efficiently removed from the dialysate by commercially available sorbent technology. Spent peritoneal dialysis fluid can be cleared of β2M when circulated against sorbent-regenerated dialysate using a high-flux membrane.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.