E3 Ubiquitin Ligase Siah2 Research Articles

Synaptic requiem: a duet for Piccolo and Bassoon

EMBO J (2013) 32:11, 954–969 doi:10.1038/emboj.2013.27; published online February122013 Neurotransmission in the brain critically depends on the maintenance of synapses as well as on regulated synaptic protein turnover. How synaptic proteostasis is held in check has remained largely enigmatic. A new paper in The EMBO Journal reports that the active zone proteins Piccolo and Bassoon put a brake on presynaptic protein turnover by restraining the activity of the E3 ubiquitin ligase Siah1, thereby preventing neurodegeneration.

The E3 Ubiquitin Ligase Siah2 Contributes to Castration-Resistant Prostate Cancer by Regulation of Androgen Receptor Transcriptional Activity

Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.

Abstract P291: The Ubiquitin Ligase Siah2 in Cardiac Response to Ischemia

Background: The E3 ubiquitin ligases SIah1 and 2 mediate a cellular response to hypoxia through its control of prolyl-hydroxylases and consequently HIF1α stability. Given the impact of hypoxia on heart function under stress, we have evaluated the effect of genetic deletion of Siah1 and 2 on cardiac function in response to myocardial infarction. Objective: To determine the physiological significance of the ubiquitin ligase Siah genes under in vivo ischemia condition. Materials and methods: To substantiate the role of Siah2 in ischemia-induced cell death, we tested difference in the response of myocardial tissue to ischemia using Siah1a +/− /Siah2 −/− mutant mice and their wildtype littermates. Analysis of heart tissue from Siah1a +/− /Siah2 −/− mice subjected to a model of 24 hours anterior descending coronary artery occlusion revealed their resistance to ischemic injury when compared with their littermates. Cardiac function, measured as EF %, was significantly less impaired in the mutant mice. Heart tissue obtained from Siah1a +/− /Siah2 −/− mice subjected to myocardial ischemia, exhibited a significantly lower degree of apoptosis (Tunel Staining), and myocardial infarct size (Evans blue, TTC). Conclusions: Collectively, these data establish that reduced expression of Siah1/2 exerts a cardioprotective function under ischemic conditions. The underlying molecular mechanisms linking cardioprotection to ischemic challenge will be discussed.

Differential regulation of PML–RARα stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1

The ubiquitin proteasome system plays an important role in normal and malignant hematopoiesis and relies on the concerted action of three enzyme families. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins. One such protein is the leukemia fusion protein PML–RARα (promyelocytic leukemia–retinoic acid receptorα) that is associated with acute promyelocytic leukemia. A limited number of UBCH8 interaction partners that participate in the UBCH8-dependent depletion of cancer-relevant proteins are known. We report here that TRIAD1 (two RING fingers and DRIL [double RING finger linked] 1), an E3 ubiquitin ligase relevant for the clonogenic growth of myloid progenitors, binds UBCH8 as well as PML–RARα. Moreover, there is concurrent induction of TRIAD1 and UBCH8 upon combinatorial treatment of acute promyelocytic leukemia cells with the pro-apoptotic epigenetic modulator valproic acid and the differentiation inducing agent all-trans retinoic acid. However, in sharp contrast to SIAH1/SIAH2 and UBCH8, TRIAD1 binding to PML–RARα has no effect on its turnover. In summary, our data exclude TRIAD1 as crucial regulator of the leukemic determinant PML–RARα, but highlight the prominence of the UBCH8/SIAH axis in PML–RARα degradation.

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.

Possible role of death receptor-mediated apoptosis by the E3 ubiquitin ligases Siah2 and POSH

BackgroundA functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. The ability of proteasome inhibitors, such as Velcade, to promote extrinsic apoptotic effects illustrates the importance of the ubiquitin proteasome system in the regulation of death receptor signaling. Here, we set out to define the UPS machinery, particularly the E3 ubiquitin ligases, that repress apoptosis through the extrinsic pathway. A cell-based genome-wide E3 ligase siRNA screen was established to monitor caspase-8 activity following the addition of TRAIL.ResultsData from the high-throughput screen revealed that targeting the RING-finger containing E3 ligase Siah2 as well as the signaling platform molecule POSH (SH3RF1) conferred robust caspase-8 activation in response to TRAIL stimulus. Silencing Siah2 or POSH in prostate cancer cells led to increased caspase activity and apoptosis in response to both TRAIL and Fas ligand. The E3 activity of Siah2 was responsible for mediating apoptosis resistance; while POSH protein levels were critical for maintaining viability. Further characterization of Siah2 revealed it to function downstream of early death receptor events in the apoptotic pathway. The observed apoptosis resistance provides one biological explanation for the induction of Siah2 and POSH reported in lung and prostate cancer, respectively. Expanding on an initial yeast-two-hybrid screen we have confirmed a physical interaction between E3 ligases Siah2 and POSH. Utilizing a yeast-two-hybrid mapping approach we have defined the spacer region of POSH, more specifically the RPxAxVxP motif encompassing amino acids 601-607, to be the site of Siah2 binding.ConclusionsThe data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.

Nuclear expression of the ubiquitin ligase seven in absentia homolog (SIAH)-1 induces proliferation and migration of liver cancer cells

Differential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis. SIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed. In HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients. Nuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth.

An Upregulation of SIAH1 After Spinal Cord Injury in Adult Rats

E3 ubiquitin ligase SIAH1 is a protein associated with the onset of nontumorigenicy in revertant tumorigenic cell lines and with several apoptotic processes. However, its role in the injury of the central nervous system remains unknown. In this study, we performed acute spinal cord injury (SCI) in adult rats and investigated the protein expression and cellular localization of SIAH1 in the spinal cord. Western blot analysis revealed that SIAH1 was low expressed in normal spinal cord. It increased at 8 h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that SIAH1 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased after SCI. Double immunofluorescence staining showed that SIAH1 was coexpressed with NeuN (neuronal marker), CNPase (oligodendroglial marker), GFAP (astroglial marker), and CD11b (microglial marker) at 1 day post-injury and was also coexpressed with active caspase-3 in neurons and glial cells after injury. In addition, double immunofluorescence staining indicated that p-c-Jun NH2-kinase (JNK) coexpressed with SIAH1 in neurons and glial cells. Coimmunoprecipitation further showed that p-JNK and SIAH1 precipitated with each other in the damaged spinal cord. Taken together, these data suggest SIAH1 involvement in the injury response of the adult spinal cord of the rats.

Abstract 3199: p53 represses TRF2 through E3 ubiquitin ligase Siah-1: Feedback regulation in telomere-initiated damage signaling

Abstract p53 plays critical roles in tumor suppression, stem cell functions and aging in vivo. The telomere-capping protein complex (shelterin) prevents functional telomeres from undergoing erosion or end-to-end fusion and from initiating unwanted DNA damage response. Uncapped, dysfunctional telomeres at the end of cellular replicative lifespan lose this protective mechanism and trigger telomere-initiated DNA damage signaling to activate p53 and thereby induce cellular senescence. Here we report that p53 in turn controls a component of the shelterin complex, TRF2, through Siah-1, a p53-inducible E3 ubiquitin ligase. Endogenous TRF2 and Siah-1 were repressed and induced, respectively, in normal human fibroblasts at replicative senescence, when p53 was physiologically activated. Spontaneous allelic loss, shRNA-mediated knockdown, dominant-negative inhibition, nutlin-3a activation and overexpression of p53 all showed that p53 induced Siah-1 and repressed TRF2. TRF2 was subject to proteasomal degradation in a p53-dependent manner. Anti-TRF2 antibody-immunoprecipitated proteins were found to undergo p53- and Siah-1-mediated ubiquitination. In vitro and in vivo ubiquitination experiments showed that the E3 ligase activity of Siah-1 was responsible for TRF2 ubiquitination. Biologically, Siah-1 knockdown or TRF2 overexpression delayed the onset of replicative senescence, suggesting that the proteolytic control of TRF2 cooperates with the transcriptional regulation of p53 target genes (e.g., p21WAF1 and microRNA-34a) to regulate p53-mediated replicative senescence. This study reveals that p53, which is a downstream effector of the DNA damage signaling from uncapped telomeres, also functions upstream to regulate the telomere-capping complex, and suggests that the p53-Siah-1-TRF2 pathway takes an integral part in orchestrating the DNA damage response at telomeres. Given that TRF2 inhibits a p53-activating kinase ATM at telomeres, a positive feedback loop involving TRF2, ATM and p53 may function to amplify DNA damage-induced and p53-mediated cellular responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3199.

Siah-1 Protein Is Necessary for High Glucose-induced Glyceraldehyde-3-phosphate Dehydrogenase Nuclear Accumulation and Cell Death in Müller Cells

The translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the nucleus has closely been associated with cell death induction. However, the mechanism of this process has not been completely understood. The E3 ubiquitin ligase siah-1 (seven in absentia homolog 1) has recently been identified as a potential shuttle protein to transport GAPDH from the cytosol to the nucleus. Previously, we have demonstrated that elevated glucose levels induce GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study investigated the role of siah-1 in high glucose-induced GAPDH nuclear translocation and subsequent cell death in retinal Müller cells. High glucose significantly increased siah-1 expression within 12 h. Under hyperglycemic conditions, siah-1 formed a complex with GAPDH and was predominantly localized in the nucleus of Müller cells. siah-1 knockdown using 50 nm siah-1 small interfering RNA significantly decreased high glucose-induced GAPDH nuclear accumulation at 24 h by 43.8 +/- 4.0%. Further, knockdown of siah-1 prevented high glucose-induced cell death of Müller cells potentially by inhibiting p53 phosphorylation consistent with previous observations, indicating that nuclear GAPDH induces cell death via p53 activation. Therefore, inhibition of GAPDH nuclear translocation and accumulation by targeting siah-1 promotes Müller cell survival under hyperglycemic conditions.

Abstract 5802: Ischemia-induced Angiogenesis is Impaired in Aminopeptidase-A deficient Mice with Decreased Stability of HIF-1 α

Background: Aminopeptidase A (APA) is a membrane-bound metalloprotease degreading bioactive peptidase by cleaving from N-terminal amino acids with several functions in angiogenesis. To assess whether APA regulates ischemia-induced angiogenesis, we explored the mechanism by which APA affects angiogenesis under hypoxia conditions and evaluated the process of perfusion recovery in the hindlimb ischemia model of APA knock-out mice (APAKO). Methods and Results: Western blotting analysis and immunostaining of endothelial cells (ECs) from mouse aorta revealed that the accumulation HIF-1 α protein and nuclear translocation in response to hypoxic challenge (1%O 2 , 12h) were blunted in APAKO (HIF-1 α / β -Actin ratio, C57BL/6J (WT) vs APAKO: 0.987±0.069 vs 0.467±0.063, mean±SE, p=0.018). Although the levels of E3 ubiquitin ligase Siah2 and prolyl-hydroxylase 3 (PHD3), both upstream regulators of HIF-1 α , did not alter under hypoxia (1%O 2 , 6h), proteasome inhibitor MG-132 restored the decreased accumulation of HIF-1 α in ECs from APAKO similar to WT. Tube formation assay under hypoxia showed ECs of APAKO formed significantly less endothelial network (Tube length, mm/field, WT vs APAKO: 35.2±3.4 vs 27.6±5.0, p=0.024), and this was partly reversed by VEGF (50ng/mL) treatment (31.2±4.8, p=0.048), but not by soluble APA peptide supplementation (26.6±4.3, p=NS). APAKO and WT mice were subjected to unilateral hindlimb ischemia. Laser Doppler perfusion imaging demonstrated decreased blood flow recovery in APAKO, (Ischemic/Non ischemic limb, WT vs APAKO: 0.80±0.07 vs 0.70±0.04, at 4 weeks, p=0.046). Tissue capillary density at 2 weeks was significantly decreased in APAKO (ECs/Muscle Ratio, WT vs APAKO: 1.57±0.15 vs 1.25±0.12; p=0.039). FACS analysis of endothelial progenitor cells revealed no significant difference between in APAKO and WT mice, suggesting that vasculogenesis was minimally involved in the reparative process from ischemia in APAKO mice. Conclusion: Ischemia-induced angiogenesis is impaired in APAKO mice due to decreased stability of HIF-1 α with upregulation of proteasomal degradation under hypoxic conditions and subsequent shortage of growth factor secretion. APA would be a functional target of ischemia-induced angiogenesis.

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-kappaB 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-1alpha 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.

Stabilization of HIPK2 by escape from proteasomal degradation mediated by the E3 ubiquitin ligase Siah1

Homeodomain-interacting protein kinase 2 (HIPK2) induces apoptosis and, thus, is maintained at a low level via ubiquitin-mediated proteolysis. In a yeast two-hybrid screen, we identified Siah1, a RING finger E3 ubiquitin ligase, as an interacting protein of HIPK2. Siah1 targeted HIPK2 for poly-ubiquitination-mediated proteasomal degradation. Degradation of HIPK2 by Siah1 was blocked by forced expression of either Mixed Lineage Kinase-3 or Epstein-Barr viral protein LMP-1, as well as by DNA damaging stimuli. These findings effectively illustrate the regulatory mechanisms underlying HIPK2 stabilization by escape from Siah1-mediated degradation, and that Siah1 is an integration target for several internal or external stimuli for HIPK2 stabilization.

Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR

The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.

Slow Breathing

The mitochondrial scaffolding protein A-kinase anchor protein 121 (AKAP121) assembles a signaling complex that includes components of the cAMP (adenosine 3',5'-monophosphate) and Src signaling pathways, facilitating their stimulation of mitochondrial metabolism. Carlucci et al . identified the E3-ubiquitin ligase Siah2 as an AKAP121 binding partner in a yeast two-hybrid screen and confirmed the interaction through in vitro assays, coimmunoprecipitation from human embryonic kidney (HEK) 293 cell lysates, and immunostaining of mouse fibroblasts. Cotransfection of HEK293 cells with AKAP121 increased association of a Siah2 mutant that lacked E3-ubiquitin ligase activity (Siah2 rm ) with mitochondria, and cotransfection with Siah2 (but not Siah2 rm ) markedly decreased AKAP121 abundance. Hypoxia promotes Siah2 transcription, and oxygen and glucose deprivation (OGD) stimulated a Siah2-dependent decrease in the abundance of AKAP121 transfected into HEK293 cells and of endogenous AKAP121 in cultured rat hippocampal neurons and mouse fibroblasts. AKAP121 was a substrate for Siah2 ubiquitination in vitro, and Siah2 overexpression promoted the ubiquitination and proteasomal degradation of AKAP121 in HEK293 cells, as did OGD. When transfected into HEK293 cells, AKAP121 increased mitochondrial membrane potential (ΔΨ m ) and metabolic activity and opposed decreases in ΔΨ m and mitochondrial metabolic activity produced by OGD; Siah2 decreased basal ΔΨ m and metabolic activity and opposed the effects of AKAP121. Further, siRNA directed against Siah2 increased metabolic activity in cells exposed to OGD. Occlusion of the middle cerebral artery in rats (which causes ischemic damage to the frontal cortex) led to a decrease in AKAP121 immunoreactivity in neurons in the ischemic region, which was blocked by siRNA directed against Siah2. The authors thus conclude that degradation of AKAP121 by Siah2 provides a mechanism whereby mitochondrial respiration can be slowed under hypoxic conditions. A. Carlucci, A. Adornetto, A. Scorziello, D. Viggiano, M. Foca, O. Cuomo, L. Annunziato, M. Gottesman, A. Feliciello, Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia. EMBO J. 27 , 1073-1084 (2008). [PubMed]

E3 ubiquitin ligase SIAH1 mediates ubiquitination and degradation of TRB3

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-κB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-β signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-β signaling.

High-resolution structure of humanD-glyceraldehyde-3-phosphate dehydrogenase

GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) is a multifunctional protein that is a target for the design of antitrypanosomatid and anti-apoptosis drugs. Here, the first high-resolution (1.75 Angstroms) structure of a human GAPDH is reported. The structure shows that the intersubunit selectivity cleft that has been leveraged in the design of antitrypanosomatid compounds is closed in human GAPDH. Modeling of an anti-trypanosomatid GAPDH inhibitor in the human GAPDH active site provides insights into the basis for the observed selectivity of this class of inhibitor. Moreover, the high-resolution data reveal a new feature of the cleft: water-mediated intersubunit hydrogen bonds that assist closure of the cleft in the human enzyme. The structure is used in a computational ligand-docking study of the small-molecule compound CGP-3466, which inhibits apoptosis by preventing nuclear accumulation of GAPDH. Plausible binding sites are identified in the adenosine pocket of the NAD(+)-binding site and in a hydrophobic channel located in the center of the tetramer near the intersection of the three molecular twofold axes. The structure is also used to build a qualitative model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. The model suggests that the convex surface near GAPDH Lys227 interacts with a large shallow groove of the Siah1 dimer. These results are discussed in the context of the recently discovered NO-S-nitrosylation-GAPDH-Siah1 apoptosis cascade.

Glycogen Synthase Kinase 3β Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH: IMPLICATIONS FOR PROTEASOMAL FUNCTION AND LEWY BODY FORMATION

alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.

Interaction of AF4 Wildtype and AF4•MLL Fusion Protein with SIAH Proteins: Indication for T(4;11) Pathobiology?.

The human AF4 gene (4q21) is recurrently involved in recipro cal translocations to the MLL gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Due to the constant presence of the derivative(11) product, the hypothesis was posed that only MLL•TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while some other MLL fusions failed. Here, we demonstrate growth transforming potential of AF4 wildtype and the AF4•MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4•MLL fusion protein against proteosomal degradation. Supported by grant Ma 1786/4-1 from the DFG.