Person:

North, Brian

CYLD negatively regulates the NF-κB signaling pathway and osteoclast differentiation largely through antagonizing TNF receptor-associated factor (TRAF)-mediated K63-linkage polyubiquitination in osteoclast precursor cells. CYLD activity is controlled by IκB kinase (IKK), but the molecular mechanism(s) governing CYLD protein stability remains largely undefined. Here, we report that SCFβ-TRCP regulates the ubiquitination and degradation of CYLD, a process dependent on prior phosphorylation of CYLD at Ser432/Ser436 by IKK. Furthermore, depletion of β-TRCP induced CYLD accumulation and TRAF6 deubiquitination in osteoclast precursor cells, leading to suppression of RANKL-induced osteoclast differentiation. Therefore, these data pinpoint the IKK/β-TRCP/CYLD signaling pathway as an important modulator of osteoclastogenesis.

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCFFbw7. DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1δ. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development.

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD+ levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

Tumor cell proliferation, survival and migration are regulated by the deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), a tumor suppressor that serves as a scaffold protein for H-Ras and TRAF2. Importantly, the oncogenic histone methyl-transferase EZH2 epigenetically down-regulates DAB2IP in a variety of tumors. Recently, we demonstrated that DAB2IP is negatively regulated by Akt-dependent phosphorylation and SCFFbw7-mediated degradation. Here, we further identify the oncoprotein Smurf1, an E3-ubiquitin ligase, as a novel negative regulator of DAB2IP. Smurf1-mediated cellular proliferation and migration are largely dependent on the presence of DAB2IP, suggesting that DAB2IP is a key effector molecule of Smurf1 oncogenic function. Additionally, we identify that similar to DAB2IP, Smurf1 is also a target of phosphorylation by both Akt1 and Akt2 kinases, which enhances Smurf1 abundance, leading to a reduction in DAB2IP. Given the role of DAB2IP in tumorigenesis and metastasis, our data identify Smurf1 as an upstream oncogenic factor that negatively regulates DAB2IP to govern aberrant cell growth and migration.

The histone deacetylase 6 (HDAC6) plays critical roles in human tumorigenesis and metastasis. As such, HDAC6-selective inhibitors have entered clinical trials for cancer therapy. However, the upstream regulator(s), especially ubiquitin E3 ligase(s), responsible for controlling the protein stability of HDAC6 remains largely undefined. Here, we report that Cullin 3SPOP earmarks HDAC6 for poly-ubiquitination and degradation. We found that the proteasome inhibitor MG132, or the Cullin-based E3 ligases inhibitor MLN4924, but not the autophagosome-lysosome inhibitor bafilomycin A1, stabilized endogenous HDAC6 protein in multiple cancer cell lines. Furthermore, we demonstrated that Cullin 3-based ubiquitin E3 ligase(s) primarily reduced the stability of HDAC6. Importantly, we identified SPOP, an adaptor protein of Cullin 3 family E3 ligases, specifically interacted with HDAC6, and promoted its poly-ubiquitination and subsequent degradation in cells. Notably, cancer-derived SPOP mutants disrupted their binding with HDAC6 and thereby failed to promote HDAC6 degradation. More importantly, increased cellular proliferation and migration in SPOP-depleted HCT116 colon cancer cells could be partly reversed by additional depletion of HDAC6, suggesting that HDAC6 is a key downstream effector for SPOP tumor suppressor function. Together, our data identify the tumor suppressor SPOP as an upstream negative regulator for HDAC6 stability, and SPOP loss-of-function mutations might lead to elevated levels of the HDAC6 oncoprotein to facilitate tumorigenesis and metastasis in various human cancers.