CONTRIBUTION OF A SPERM PROTEIN, PAWP, TO THE SIGNAL TRANSDUCT PATHWAY DURING VERTEBRATE FERTILIZATION

PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early and later egg activation events including meiotic resumption, pronuclear formation and egg cleavage. Based on the fact that calcium increase is universally accepted as the sole requirement for egg activation, we hypothesized that PAWP is an upstream regulator of the calcium signaling pathway during fertilization. Intracellular calcium increase was detected by two-photon laser scanning fluorescence microscopy following microinjection of recombinant PAWP into Xenopus oocytes, bolstering our hypothesis and suggesting the involvement of a novel PAWP-mediated signaling pathway during fertilization. The N-terminal of PAWP shares a high homology to WW domain binding protein while the C-terminal half contains a functional PPXY motif, which allows it to interact with group I WW domain proteins. These structural considerations together with published data indicating that PPXY synthetic peptide derived from PAWP inhibits ICSI-induced fertilization led to the hypothesis that PAWP triggers egg activation by binding to a group I WW domain protein in the oocyte. By far-Western analysis of oocyte cytoplasmic fraction, PAWP was found to bind to a 52 kDa protein. The competitive inhibition studies with PPXY synthetic peptide, WW domain constructs, and their point mutants demonstrated that the interaction between PAWP and its binding partner is specifically via the PPXY-WW domain module. The 52 kDa protein band crossreacted with antibodies against group I WW domain protein YAP in Western blot assay, indicating that this 52 kDa PAWP binding partner is either YAP or a YAP-related protein. In addition, the far-Western competitive inhibition studies with recombinant GST fusion protein YAP and another WW domain-containing protein, TAZ, demonstrated that the binding of PAWP to its binding partner was significantly reduced by TAZ, providing evidence that TAZ could be the 52 kDa protein candidate. Mass spectrometry was employed to identify this PAWP binding partner candidate. However, due to the low abundance of the candidate protein and the complexity of the sample, several strategies are still needed to enrich this protein. This study correlates PAWP induced meiotic resumption and calcium efflux at fertilization and uncovers a 52 kDa candidate WW domain protein in the oocyte cytoplasm that most likely interacts with PAWP to trigger egg activation.

Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

THE ROLE OF LIPOPROTEIN(a)/APOLIPOPROTEIN(a) IN ENDOTHELIAL DYSFUNCTION: MECHANISTIC STUDIES IN VASCULAR ENDOTHELIUM

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis.

Novel interaction between RET and membrane cytoskeletal-linker protein ezrin

RET is a receptor tyrosine kinase that mediates key signaling events, and promotes cell survival, development, and migration. Activation of RET requires a ligand from the glial cell line-derived neurotrophic factor (GDNF) family and a co-receptor from the GDNF family receptor α (GFRα). Alternative splicing of RET leads to two major isoforms, RET9 and RET51, that contain distinct C-terminal amino acids. Differences in their cytoplasmic tails confer differential binding to adaptor proteins, and in this study, the membrane cytoskeletal-linker protein ezrin was shown in an interaction with RET51, but not RET9, in a ligand- and kinase-dependent manner. Results indicated that Y1096 on RET51 is the ezrin recruitment site, and the adaptor protein Grb2 may mediate this interaction. These results suggest that ezrin may play a role in the downstream signaling and recycling pathways of RET51. Thus, the identified novel interaction may provide insight in the longer term into how ezrin and RET51 contribute together to functional processes such as cell migration and invasion.