The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The ␣-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-␣ (including HIF-1␣ and HIF-2␣). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-␣ is required for the interaction between HIF and VHL. It has previously been established that cobalt mimics hypoxia and causes accumulation of HIF-1␣ and HIF-2␣. However, little is known about the mechanism by which this occurs. In an earlier study, we demonstrated that cobalt binds directly to the ODD domain of HIF-2␣. Here we provide the first evidence that cobalt inhibits pVHL binding to HIF-␣ even when HIF-␣ is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2␣ that are required for pVHL binding prevented the binding of cobalt and stabilized HIF-2␣ during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-␣ and thereby preventing the degradation of HIF-␣.Hypoxia is a critical stimulus in many physiological and disease states (1). Cells respond to hypoxia by regulating the expression of a number of genes, including erythropoietin, vascular endothelial growth factor, and various glycolytic enzymes (2-5). This regulation is mediated in part by transcription factors of the hypoxia-inducible factor (HIF) 1 family (6). HIF-1␣ and HIF-2␣ are basic helix-loop-helix Per-Arnt-Sim (PAS) domain proteins (7) that form a heterodimer with the aryl hydrocarbon nuclear receptor translocator protein. Previous studies have shown that HIF-1␣ protein accumulates rapidly during hypoxia without a significant increase in HIF-1␣ mRNA levels (8). HIF-2␣, which is also known as endothelial PAS domain protein-1, shares close sequence and structural homology with HIF-1␣ (9). Like HIF-1␣, the levels of HIF-2␣ protein are low during normoxia and accumulate when cells are exposed to hypoxia, proteasomal inhibitors, transition metals (e.g. cobalt), iron chelators, or reducing agents (10). During normoxia, the HIF-␣ (HIF-1␣ and HIF-2␣ are referred to here simply as HIF-␣, except where noted otherwise) proteins are continuously degraded by ubiquitin-and proteasome-dependent pathway. Detailed studies of HIF-␣ proteins revealed a 200-amino acid sequence, called the oxygen-dependent degradation domain (ODD) that is responsible for its degradation in the presence of oxygen (11,12). The von Hippel-Lindau (pVHL) protein, a tumor suppressor protein, mediates the ubiquitination and degradation of HIF-␣ by binding to the ODD domain under conditions of normoxia (13,14). Recent findings revealed that pVHL-mediated degradation requires hydroxylation of specific proline residues within the ODD (15-18). The hydroxylation of these proline residues may be critical for regulating the HI...

BackgroundAlthough overexpression of nitric oxide synthases (NOSs) has been found associated with prostate diseases, the underlying mechanisms for NOS-related prostatic diseases remain unclear. One proposed mechanism is related to the S-nitrosylation of key regulatory proteins in cell-signaling pathways due to elevated levels of NO in the prostate. Thus, our primary objective was to identify S-nitrosylated targets in an immortalized normal prostate epithelial cell line, NPrEC.Methodology/Principal FindingsWe treated NPrEC with nitroso-cysteine and used the biotin switch technique followed by gel-based separation and mass spectrometry protein identification (using the LTQ-Orbitrap) to discover S-nitrosylated (SNO) proteins in the treated cells. In parallel, we adapted a peptide pull-down methodology to locate the site(s) of S-nitrosylation on the protein SNO targets identified by the first technique. This combined approach identified 116 SNO proteins and determined the sites of modification for 82 of them. Over 60% of these proteins belong to four functional groups: cell structure/cell motility/protein trafficking, protein folding/protein response/protein assembly, mRNA splicing/processing/transcriptional regulation, and metabolism. Western blot analysis validated a subset of targets related to disease development (proliferating cell nuclear antigen, maspin, integrin β4, α-catenin, karyopherin [importin] β1, and elongation factor 1A1). We analyzed the SNO sequences for their primary and secondary structures, solvent accessibility, and three-dimensional structural context. We found that about 80% of the SNO sites that can be mapped into resolved structures are buried, of which approximately half have charged amino acids in their three-dimensional neighborhood, and the other half residing within primarily hydrophobic pockets.Conclusions/SignificanceWe here identified 116 potential SNO targets and mapped their putative SNO sites in NPrEC. Elucidation of how this post-translational modification alters the function of these proteins should shed light on the role of NO in prostate pathologies. To our knowledge, this is the first report identifying SNO targets in prostate epithelial cells.

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/β-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either β-catenin (Ctnnb1 cKO ) or co-receptors Lrp5/6 (Lrp5/6 cKO ) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gainof-function mutant (Ctnnb1 cGOF ) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of β-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1 cKO . Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1 cKO mice. Furthermore, ChIP and promoter-luciferase assays show that β-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/β-catenin/ Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), α-momorcharin and β-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of β-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

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Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

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Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.