Purification of SELENOP by nickel metal affinity chromatography eliminates its susceptibility to degradation under these conditions (Figure 5, lanes 13-14). process of SELENOP uptake using a robust selenium uptake assay that measures selenium utilization in cells fed 75Se-SELENOP. Using a series of inhibitors and modulators we have identified specific regulators of the process and found that SELENOP must be in an oxidized state for uptake. This assay also demonstrates that the proposed SELENOP receptor APOER2 is not required for selenium delivery by SELENOP to cells in culture. gene that encodes SELENOP were found to have selenium deficiency in the brain and testis, especially when fed lower selenium diets[5, 6]. The link between SELENOP and the brain-testis axis is phenotypically manifested as male sterility and severe neurologic dysfunction, the latter occurring only under conditions of limited selenium supply[5, 6]. Recent studies have shown, however, that even under selenium replete conditions, mice that lack SELENOP have persistent neurologic defects[7C9]. cIAP1 Ligand-Linker Conjugates 15 hydrochloride Interestingly, SELENOP expression is absolutely required for male fertility as supplemental selenium cannot reverse the defects in sperm motility and morphology, which is due to lack of GPX4 expression[10]. Evidence suggests that SELENOP delivers selenium to cells through receptor mediated endocytosis via Megalin in the kidney[11], cIAP1 Ligand-Linker Conjugates 15 hydrochloride APOER2 in the testis and brain[12], or by pinocytosis during maternal to fetal transfer[13]. Further, inhibitor studies have implicated clathrin-dependent delivery to the lysosomes as the initial processing event for SELENOP[14], but the fate of the protein and its selenium cargo in the lysosome has not been determined. It has long been proposed that SELENOP is degraded and that the resulting Sec residues are metabolized by Scly[15]. If processing by SCLY were the primary mechanism by which selenium is released from SELENOP, then animals lacking the gene encoding should harbor a null (as they can be immunoprecipitated with an anti-SELENOP antibody[14]. Fetal bovine serum In general, the labeling of cells with radioactive selenium is performed in the absence of serum in order to minimize the non-radioactive selenium content during the uptake of 75Se. We set out to determine whether the selenium species present in fetal bovine serum were effective competitors for labeling with either 75Se-SELENOP or 75Se-selenite. Figure 4C shows that the amount of 75Se cIAP1 Ligand-Linker Conjugates 15 hydrochloride incorporated into endogenous selenoproteins in HeLa cells is the same regardless of the presence or absence of 10% serum. This data suggests that the selenium species that are present in serum are not effective competitors for selenium uptake either due to low concentrations or poor bioavailability. In vitro processing To gain insight into the mechanism by which SELENOP may be processed in cells, we examined the stability of the protein in the context of crude conditioned medium. Since evidence thus far suggests that SELENOP may be degraded in the lysosome, we analyzed the stability in the presence of acidifying and reducing conditions. Figure 5 (lanes1-12) shows that a crude preparation of 75Se-SELENOP is apparently degraded only in the presence of 0.5% acetic acid and 2 mM DTT. This result suggests the presence of an acid protease in the conditioned medium of HepG2 cells. Purification of SELENOP by nickel metal affinity chromatography eliminates its susceptibility to degradation under these conditions (Figure 5, lanes 13-14). These results indicate that SELENOP is very stable in standard conditions, but is susceptible to proteolysis by an acid protease only under acidic and reducing conditions. This may provide clues as to the conditions required to achieve efficient proteolysis of SELENOP, operating under the assumption that proteolysis is a key step in the recovery of selenium from SELENOP. Zebrafish Selenop can be taken up by human embryonic kidney cells In order to examine the specificity of SELENOP uptake, we took advantage of our ability to produce zebrafish Selenop in human embryonic kidney cells (HEK-293). We stably transfected HEK-293 cells with the cDNA encoding zebrafish SELENOP harboring a C-terminal FLAG tag, labeled these cells with 75Se-selenite and purified the radiolabeled SELENOP from the conditioned medium with anti-FLAG affinity beads, which also removed the free 75Se-selenite. Figure 6A shows the purified zebrafish SELENOP recovered after purification which was free of contaminating selenoproteins and free selenium. This preparation Adipoq was then added to naive HEK-293 cells and the labeling of endogenous selenoproteins was compared to that obtained with 75Se-selenite. Figure 6B clearly shows that HEK293 cells are able to utilize zebrafish.