The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are regulated still remain to be elucidated. We describe here a novel murine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro.

Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells (ESCs). Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating endogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells (ECCs), although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanistically, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interestingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in controlling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosagedependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.

Recognition of viral double-stranded RNA by Toll-like receptor 3 (TLR3) triggers activation of the transcription factors NF-κB and interferon regulated factor 3, leading to induction of type I interferons and proinflammatory cytokines. TIR-domain-containing adapter-inducing interferon-β (TRIF) is an adapter protein required for TLR3-mediated signaling. Here we identified the E3 ubiquitin ligase WW domaincontaining protein 2 (WWP2) as a TRIF-associated protein by biochemical purification. WWP2 mediated K48-linked ubiquitination and degradation of TRIF upon TLR3 activation. Overexpression of WWP2 inhibited TLR3-mediated NF-κB and interferon regulated factor 3 activation, whereas knockdown of WWP2 had opposite effects. We generated Wwp2-deficient mice to further investigate the roles of Wwp2 in innate immune responses. Consistently, production of IFN-β, CCL5, TNFα, and IL-6 in response to the TLR3 ligand poly(I:C) was elevated in Wwp2 −/− macrophages and Wwp2-deficient mice exhibited increased susceptibility to poly(I:C)-induced death than the control littermates. Our findings suggest that WWP2 negatively regulates TLR3-mediated innate immune and inflammatory responses by targeting TRIF for ubiquitination and degradation.T oll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors (PRRs) that are critically involved in host defense from plants to humans. So far, 13 TLRs (named TLR1 to TLR13) have been identified in humans and mice, each of which recognizes a distinct set of pathogen-associated molecular patterns (PAMPs) (1). TLRs contain an extracellular domain consisting of leucine rich repeats which is responsible for PAMP recognition, a transmembrane domain, and a conserved cytoplasmic toll/IL-1 receptor (TIR) domain which is able to mediate homotypic protein-protein interactions (2). The TIR domains of TLRs are responsible for their homo-or hetero-dimerization, and upon ligand stimulation, they also act as platforms to recruit downstream TIR domain-containing adaptor proteins and signaling molecules, leading to the activation of transcription factors such as NF-κB and interferon regulated factor 3 (IRF3) (1, 3, 4). These transcription factors act alone or in collaboration to induce transcription of proinflammatory cytokines and/or type I interferons (IFNs).Among the TLRs, TLR3 has been reported to recognize viral dsRNA as well as its analog poly(I:C). Recognition of these ligands by TLR3 activates signaling pathways leading to the activation of NF-κB and IRF3 and subsequent production of type I IFNs and proinflammatory cytokines (5). TLR3-mediated signaling critically depends on the TIR domain-containing adapter TIRdomain-containing adapter-inducing interferon-β (TRIF; also called TICAM-1) (6). It has been shown that Trif −/− lung fibroblasts are defective in poly(I:C)-induced activation of NF-κB and IRF3 as well as production of type I IFNs, demonstrating that TRIF is indispensable for TLR3-mediated signaling (7). In addition, the TLR4 ligand LPS has been shown to signal through TRIF-...

No abstract

In this article, we report the enhanced anti‐aging and mechanical properties of waterborne polyurethanes (WPUs) modified by lignin amines, which were prepared from lignins, formaldehyde, and diethylenetriamine via a Mannich reaction. When we introduced amino groups into the lignin, its solubility in water and reactivity with isocyanate was obviously improved. Consequently, the interaction between WPU and the lignin additive were promoted; this provided a higher mechanical performance, that is, a higher tensile strength and elongation at break. More importantly, the intrinsic antioxidative activity of lignin also benefitted the modified WPU in terms of aging resistance. According to artificial accelerated aging tests, mechanical measurement, attenuated total reflectance–Fourier transform infrared spectroscopy, and scanning electron microscopy analysis, the lignin amine modified WPU did not show any evidence of degradation after 2 months of artificial accelerated aging, whereas the pure WPU was obviously degraded. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 1736–1742, 2013

POU domain transcription factor Oct-4 plays a crucial role in maintaining self-renewal and pluripotency of embryonic stem (ES) cells in a concentration-dependent manner. However, the molecular mechanism controlling Oct-4 levels in ES cells remains largely unknown. To explore the molecular mechanism regulating Oct-4 function, we constructed a mouse ES cell cDNA library and performed yeast two-hybrid screening using the POU domain of Oct-4 as bait. Here, we present novel evidence for Oct-4 interaction with Ubc9, an E2 conjugation enzyme for SUMO modification, and its modification by SUMO-1. The SUMO acceptor site was identified at lysine residue 118. Importantly, disruption of Oct-4 sumoylation reduced Oct-4 protein stability and self-renewal capacity in ES cells. Interestingly, expression of cYes was found to reduce when Oct-4 sumoylation was disrupted or Oct-4 expression downregulated in ES cells. We further demonstrate that Oct-4 was recruited to the cYes promoter region, suggesting that cYes might be a novel downstream gene of Oct-4. Taken together, we first demonstrate the post-translational modification of endogenous Oct-4 by SUMO and the role of sumoylation in regulating Oct-4 protein stability and function. Our findings provide new evidence for the important role of post-translational modification in controlling Oct-4 function in ES cells.

No abstract

Wood fiber of aspen was used as a reinforced filler in linear low-density polyethylene (LLDPE). To improve the compatibility between the wood fiber and the LLDPE matrix, the wood fiber was treated with titanate coupling agents (i.e., TC-PBT and TC-POT) or grafted by acrylonitrile. Both treatments resulted in an improvement in the mechanical properties of the resultant composites compared with the composites filled with the untreated wood fiber. Moreover, the grafting method displayed a more obvious benefit than that of titanate coupling methods to the mechanical property improvement. This was attributed to the crystalline structure of the wood fiber to be destroyed by grafting acrylonitrile, and the amorphous fiber was easily deformed to enhance fiber adhesion at the LLDPE matrix. In addition, the effect of the concentration of the filled wood fiber and the amount of coupling agent or grafting ratio on the mechanical properties of composites are discussed.