In mammalian cells, Siah proteins can polyubiquitylate several seemingly unrelated proteins, with Siah1 and Siah2 having overlapping functions. In Drosophila, SINA has been shown to cooperate with Phyllopod (PHYL), Ebi and UBCD1 to facilitate the ubiquitylation and degradation of the transcriptional corepressor tramtrack 88 (TTK88) ( Li et al., 1997 Tang et al., 1997 Boulton et al., 2000). Drosophila Seven in Absentia (SINA) and mammalian Seven in Absentia Homolog (Siah) are RING-containing proteins that function in protein degradation as ubiquitin ligases. Specificity is achieved by ubiquitin ligases recognizing specific degradation signals or ‘degrons’ ( Laney and Hochstrasser, 1999). The specificity of the process is defined by the final step in which a ubiquitin ligase, either alone or as part of a complex, transfers ubiquitin to the substrate protein ( Glickman and Ciechanover, 2002). Protein ubiquitylation is a three-step, enzymatic process ( Hochstrasser, 2000). Recently, we and others have shown that, under hypoxic conditions, members of the Siah ubiquitin ligase family can polyubiquitylate and target PHD and FIH for proteasomal degradation ( Nakayama et al., 2004, 2007 Khurana et al., 2006 Fukuba et al., 2007), thus stabilizing Hif-1α. This hydroxylation prevents the interaction between Hif-1α and transcriptional co-activators CBP/p300 ( Lando et al., 2002). Furthermore, Hif-1α is hydroxylated at Asn803 by the asparaginyl hydroxylase factor-inhibiting Hif-1 (FIH) ( Lando et al., 2002). After being polyubiquitylated, Hif-1α is degraded by the 26S proteasome ( Salceda and Caro, 1997 Huang et al., 1998 Maxwell et al., 1999). The Pro402/Pro564 hydroxylated Hif-1α protein has increased affinity for the E3 ubiquitin-protein ligase complex composed of the von Hippel–Lindau tumor suppressor protein VHL, elongin B and C and cullin 2. Under normoxia, Hif-1α is hydroxylated at two conserved proline residues (Pro402 and Pro564) by prolyl-hydroxylases (PHDs) ( Epstein et al., 2001 Jaakkola et al., 2001). In the Hif-1 complex, the Hif-1α subunit is constitutively present within the cell, whereas Hif-1β is stabilized under hypoxic conditions ( Semenza, 1999). Hif-1 consists of two subunits, Hif-1α and Hif-1β. The key transcription factor for the hypoxic response pathways is hypoxia-inducible factor 1 (Hif-1) ( Iyer et al., 1998 Semenza, 2000 Schofield and Ratcliffe, 2004).
The main cellular response to hypoxic stress is the upregulation of hypoxia-responsive genes, including VEGF, Glut-1, and CA9 ( Semenza, 1999). Solid tumor growth is associated with areas of poor oxygen supply within the tumor mass. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.
In addition, levels of Hif-1α and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1α protein levels and Hif-1-mediated gene expression under hypoxia. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. The hydroxylation of Hif-1α by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. Unexpectedly he falls in love with Reyyan.Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1α (Hif-1α), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Miran is seeking revenge for his parents death so he plans to marry daughter Reyyan from the family who were responsible for that.
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