Interaction and localization of the retinitis pigmentosa protein RP2 and NSF in retinal photoreceptor cells.

RP2 is a ubiquitously expressed protein encoded by a gene associated with X-linked retinitis pigmentosa (XLRP), a retinal degenerative disease that causes severe vision loss. Previous in vitro studies have shown that RP2 binds to ADP ribosylation factor-like 3 (Arl3) and activates its intrinsic GTPase activity, but the function of RP2 in the retina, and in particular photoreceptor cells, remains unclear. To begin to define the role of RP2 in the retina and XLRP, we have conducted biochemical studies to identify proteins in retinal cell extracts that interact with RP2. Here, we show that RP2 interacts with N-ethylmaleimide sensitive factor (NSF) in retinal cells as well as cultured embryonic kidney (HEK293) cells by mass spectrometry-based proteomics and biochemical analysis. This interaction is mediated by the N-terminal domain of NSF. The E138G and DeltaI137 mutations of RP2 known to cause XLRP abolished the interaction of RP2 with the N-terminal domain of NSF. Immunofluorescence labeling studies further showed that RP2 colocalized with NSF in photoreceptors and other cells of the retina. Intense punctate staining of RP2 was observed close to the junction between the inner and outer segments beneath the connecting cilium, as well as within the synaptic region of rod and cone photoreceptors. Our studies indicate that RP2, in addition to serving as a regulator of Arl3, interacts with NSF, and this complex may play an important role in membrane protein trafficking in photoreceptors and other cells of the retina.

Oviductal glycoprotein (OVGP1, MUC9): a differentiation-based mucin present in serum of women with ovarian cancer.

INTRODUCTION: Epithelial ovarian carcinomas are highly lethal because most are detected at late stages. A previous immunohistochemical analysis showed that oviductal glycoprotein 1 (OVGP1), a secretory product of the oviductal epithelium under estrogen dominance, is produced predominantly by borderline and low-grade malignant epithelial ovarian tumors. In the present study, we investigated OVGP1 as a possible serum marker for the detection of ovarian cancer. METHODS: We generated a highly specific monoclonal antibody, clone 7E10, to human OVGP1. Using 7E10 and a polyclonal antibody, a sandwich enzyme-linked immunosorbent assay was developed to assay OVGP1 levels in 135 normal sera, and sera from 21 benign tumors, 12 borderline tumors, and 87 ovarian cancers (18, grade 1-2 serous; 44, grade 3 serous; 10, mucinous; 10, clear cell; and 5, endometrioid). RESULTS: Using a 95% confidence interval cutoff from the mean of normal postmenopausal sera, median OVGP1 levels were elevated in the sera from 75% of the women with borderline tumors and 80% of the women with mucinous, 60% with clear cell, 59% with grade 1 and 2 serous, 22% with grade 3 serous, and 0% with endometrioid carcinomas. By stage, OVGP1 levels were highest in the sera from the borderline tumors, stage I and II serous carcinomas, and mucinous carcinomas. OVGP1 levels varied independently of cancer antigen 125 (CA125). CONCLUSIONS: Increases in OVGP1 serum levels vary with ovarian tumor histotypes and stages. Being differentiation based, OVGP1 seems to detect a different spectrum of ovarian epithelial cancers than other markers and thus should be a useful adjunct for more accurate detection, particularly of early serous ovarian cancers and mucinous carcinomas, which tend to lack increased CA125.

Proteomic Analysis and Functional Characterization of P4-ATPase Phospholipid Flippases from Murine Tissues.

P4-ATPases are a subfamily of P-type ATPases that flip phospholipids across membranes to generate lipid asymmetry, a property vital to many cellular processes. Mutations in several P4-ATPases have been linked to severe neurodegenerative and metabolic disorders. Most P4-ATPases associate with one of three accessory subunit isoforms known as CDC50A (TMEM30A), CDC50B (TMEM30B), and CDC50C (TMEM30C). To identify P4-ATPases that associate with CDC50A, in vivo, and determine their tissue distribution, we isolated P4-ATPases-CDC50A complexes from retina, brain, liver, testes, and kidney on a CDC50A immunoaffinity column and identified and quantified P4-ATPases from their tryptic peptides by mass spectrometry. Of the 12 P4-ATPase that associate with CDC50 subunits, 10 P4-ATPases were detected. Four P4-ATPases (ATP8A1, ATP11A, ATP11B, ATP11C) were present in all five tissues. ATP10D was found in low amounts in liver, brain, testes, and kidney, and ATP8A2 was present in significant amounts in retina, brain, and testes. ATP8B1 was detected only in liver, ATP8B3 and ATP10A only in testes, and ATP8B2 primarily in brain. We also show that ATP11A, ATP11B and ATP11C, like ATP8A1 and ATP8A2, selectively flip phosphatidylserine and phosphatidylethanolamine across membranes. These studies provide new insight into the tissue distribution, relative abundance, subunit interactions and substrate specificity of P4-ATPase-CDC50A complexes.