Homology modeling and in silico screening of inhibitors for the substrate binding domain of human Siah2: implications for hypoxia-induced cancers

The three-dimensional (3D) structure of the substrate binding domain (SBD) of human ubiquitin ligase Siah2 (seven in absentia homolog) was constructed based on the homology modeling approach using the Modeller 9v7 program. The molecular dynamics method was utilized to refine the model and it was further assessed by ProSA, three-dimensional structural superposition (3d-SS) and PROCHEK in order to analyze the quality and reliability of the generated model. Furthermore, we predicted the binding pocket of Siah2 and also validated it by both blind and normal docking using a known functional inhibitor, menadione. Using structure-based high-throughput virtual screening, we identified five lead drug-like molecules against the modeled SBD of Siah2 and analyzed its pharmacokinetic properties to identify the potential inhibitors for Siah2. The docking results for menadione and the lead molecules at the ligand binding site of SBD of Siah2 revealed that the residue Ser39 (corresponding to Ser167 in the full-length protein) is consistently involved in strong hydrogen bonding, and plays an important role in phosphorylation and the enhanced activity of Siah2.     

Regulation of the ring finger E3 ligase Siah2 by p38 MAPK

The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.     

Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.     

Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling.

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 A resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded beta-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-alpha receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-alpha-mediated NF-kappa B activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.     

Siah: New Players in the Cellular Response to Hypoxia

Prolyl-hydroxylation of HIF-1α is a prerequisite for pVHL binding to HIF-1α, which results in degradation of HIF-1α by the ubiquitin-proteasome pathway. Hydroxylation of HIF-1α is mediated by the family of prolyl-hydroxylase proteins (PHD). In hypoxia, HIF-1α is stabilized as a result of inhibition of HIF-1α hydroxylation, which in part is achieved by decreased activity of PHD enzymes at very low oxygen concentrations. We recently demonstrated that in hypoxia the stability of 2 of 3 PHDs (1 and 3) is regulated by the E3 ligases Siah1/2. Consequently, in hypoxia Siah determines the availability of PHD1/3, which otherwise modify HIF-1α to enable its association-dependent degradation by pVHL. These findings define a newly discovered layer in the regulation of HIF-1α in hypoxia. The roles of Siah activities in hypoxia responses are discussed.     

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF‐κB signaling pathways. Both Ras/mitogen‐activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high‐throughput electro‐chemiluninescent‐based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self‐ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF‐1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah‐KO cells, pointing to its Siah‐dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS‐independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.     

Generation and analysis of Siah2 mutant mice

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.     

Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon

Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.     

Elucidation of the substrate binding site of Siah ubiquitin ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two beta strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.     

Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP)

Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.     

USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

Isolation of Xenopus HIF-prolyl 4-hydroxylase and rescue of a small-eye phenotype caused by Siah2 over-expression

Hypoxia is an important physiological condition during embryonic development. Hypoxia-inducible factor (HIF) is the mediator of hypoxic response of cells. The prolyl hydroxylase (PHD) of HIF plays a key role in stabilizing of HIF and the oxygen homeostasis of organisms. In this study, we isolated two PHD proteins, PHD45 and PHD28, and characterized them during the embryonic development of Xenopus laevis, which is an excellent model for embryonic development because of the ease of embryonic manipulation and the feasibility of transgenesis. Based on amino acid sequences, Xenopus PHD45 and PHD28 were homologous with human PHD2 and PHD3, respectively. In embryonic development, PHD45 expression was complementary to that of PHD28. xHIF-1alpha protein level was at a maximum around stage 20 when expression of PHD45 disappeared, while expression of PHD28 reached a maximum at stage 20, suggesting that PHD28 is inducible by HIF-1alpha. Recently, Siah2 was found to be an ubiquitin ligase of PHD proteins and to regulate degradation of PHD proteins. Over-expression of xSiah2 decreased PHD45 but not PHD28 and caused the small-eye phenotype of Xenopus. Additional over-expression of PHD47 rescued the abnormality caused by xSiah2, suggesting that the level of expression or activity of PHD proteins is important to the maintenance of homeostasis in embryonic development.     

TIN2 stability is regulated by the E3 ligase Siah2

Telomeres are coated by shelterin, a six-subunit complex that is required for protection and replication of chromosome ends. The central subunit TIN2, with binding sites to three subunits (TRF1, TRF2, and TPP1), is essential for stability and function of the complex. Here we show that TIN2 stability is regulated by the E3 ligase Siah2. We demonstrate that TIN2 binds to Siah2 and is ubiquitylated in vivo. We show using purified proteins that Siah2 acts as an E3 ligase to directly ubiquitylate TIN2 in vitro. Depletion of Siah2 led to stabilization of TIN2 protein, indicating that Siah2 regulates TIN2 protein levels in vivo. Overexpression of Siah2 in human cells led to loss of TIN2 at telomeres that was dependent on the presence of the catalytic RING domain of Siah2. In contrast to RNAi-mediated depletion of TIN2 that led to loss of TRF1 and TRF2 at telomeres, Siah2-mediated depletion of TIN2 allowed TRF1 and TRF2 to remain on telomeres, indicating a different fate for shelterin subunits when TIN2 is depleted posttranslationally. TPP1 was lost from telomeres, although its protein level was not reduced. We speculate that Siah2-mediated removal of TIN2 may allow dynamic remodeling of the shelterin complex and its associated factors during the cell cycle.     

A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism

The phosphorylation of the amylopectin fraction of starch catalyzed by the alpha-glucan, water dikinase (GWD, EC 2.7.9.4) plays a pivotal role in starch metabolism. Limited proteolysis of the potato tuber (Solanum tuberosum) GWD (StGWD, 155 kDa) by trypsin primarily produced stable fragments of 33 and 122 kDa, termed the SBD fragment and N11, respectively, as generated by trypsin cleavage at Arg-286. SBD and N11 were generated using recombinant DNA technology and purified to near homogeneity. Tandem repeat sequences, SBD-1 and SBD-2, of a region that is significantly similar in sequence to N-terminal regions of plastidial alpha-amylases are located in the N-terminus of StGWD. The SBD-1 motif is located within the sequence of the SBD fragment, and our results demonstrate that the fragment composes a new and novel carbohydrate-binding module (CBM), apparently specific for plastidial alpha-glucan degradation. By mutational analyses of conserved Trp residues located within the SBD-1 motif, W62 and W117, we show that these aromatic residues are vital for carbohydrate binding. N11 still possessed starch phosphorylating activity, but with a 2-fold higher specific activity compared to that of wild type (WT) StGWD using potato starch as the glucan substrate, whereas it had double the K(m) value for the same substrate. Furthermore, investigation of the chains phosphorylated by WT StGWD and N11 shows that N11 exhibits a higher preference for phosphorylating shorter chains of the amylopectin molecule as compared to WT. From analyses of the glucan substrate specificity, we found up to 5-fold higher specific activity for N11 using amylose as the substrate.     

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.