Huibing Wang

FBXL20-mediated Vps34 ubiquitination as a p53 controlled checkpoint in regulating autophagy and receptor degradation

Vacuolar protein-sorting 34 (Vps34), the catalytic subunit in the class III PtdIns3 (phosphatidylinositol 3) kinase complexes, mediates the production of PtdIns3P, a key intracellular lipid involved in regulating autophagy and receptor degradation. However, the signal transduction pathways by which extracellular signals regulate Vps34 complexes and the downstream cellular mechanisms are not well understood. Here we show that DNA damage-activated mitotic arrest and CDK activation lead to the phosphorylation of Vps34, which provides a signal to promote its ubiquitination and proteasomal degradation mediated by FBXL20 (an F-box protein) and the associated Skp1 (S-phase kinase-associated protein-1)-Cullin1 complex, leading to inhibition of autophagy and receptor endocytosis. Furthermore, we show that the expression of FBXL20 is regulated by p53-dependent transcription. Our study provides a molecular pathway by which DNA damage regulates Vps34 complexes and its downstream mechanisms, including autophagy and receptor endocytosis, through SCF (Skp1-Cul1-F-box)-mediated ubiquitination and degradation. Since the expression of FBXL20 is regulated by p53-dependent transcription, the control of Vps34 ubiquitination and proteasomal degradation by FBXL20 and the associated SCF complex expression provides a novel checkpoint for p53 to regulate autophagy and receptor degradation in DNA damage response.

STUDY ON THE PHYSIOLOGICAL MECHANISM OF BORON UTILIZATION EFFICIENCY IN RAPE CULTIVARS

In order to uncover the mechanism of boron (B) utilization efficiency, B metabolism was studied through B forms and their balance of transformation. According to the reaction between B and some sugars in rape (Brassica napus L.), B was divided into free B, semi-bound B, and bound B using a sequential extraction procedure. One B-efficient rape cultivar (9589), one B-inefficient rape cultivar (95105), and the F1 hybrid (95105 × 9589) were involved in this pot experiment, which was carried out during 1998–1999. Two externally applied B treatments (B1: 0.05 mg kg−1 and B2: 0.25 mg kg−1) were used, and in both B treatments, the results showed that the concentration of the three B forms differed between the different cultivars. The free B and bound B concentration in the B-efficient cultivar (9589) was significantly lower than that in the B-inefficient cultivar (95105), and the F1 hybrid (95105 × 9589) was intermediate, but the semi-bound B concentration showed the opposite trend. Boron could react with some sugars which contained the vicinal group in cis position, which showed closely the relation between B and some sugar in plants. A significant difference of fructose content existed between cultivars. Using thin-layer chromatography and ultraviolet-scan analysis fructose was shown to react easily with B. From the close relationship between B and sugar, a hypothesis concerning the physiological mechanism of B utilization efficiency was formulated; a reaction constant km for the balance of B forms may indicate to some degree whether rape cultivars are B efficient.

Effect of heat treatment on the properties of Co50Ni20Ga30 ferromagnetic shape memory alloy ribbons

The structure, magnetic properties and martensitic transformation behaviour were investigated for as-spun Co50Ni20Ga30 ribbons under various heat-treatment conditions. When the as-spun ribbons were annealed at 400 degrees C, the saturation magnetization decreased from 49.5 to 40.1 emu g(-1) and the coercivity increased from 18 to 2622 Oe, accompanied by the disappearance of the martensitic transformation. Re-treating the annealed sample at different temperatures from 600 to 1200 degrees C, the martensitic transformation reoccurred and the T-M was adjusted between 132 and 249 K.

PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP

Significance This study demonstrates Pellino 1 (PELI1) as an important modulator that exerts opposite regulatory functions on apoptosis and necroptosis, two distinct forms of regulated cell death mechanisms. K63 ubiquitination of RIPK1 mediated by PELI1 depends on the kinase activity of RIPK1, which provides a mechanism to explain why inhibition of RIPK1 kinase activity by Nec-1s can also reduce its ubiquitination during necroptosis. Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1−/− mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.

Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis

Significance While the critical role of RIPK1 kinase activity in mediating necroptosis and RIPK1-dependent apoptosis has been established, we still know little about how the nonkinase domains of RIPK1 regulate its kinase activity. Establishing the role of RIPK1-death domain (DD) in mediating RIPK1 activation and formation of complex II provides an important insight into the molecular mechanism by which RIPK1 is activated during the transition from complex I to complex II. These results suggest that RIPK1-DD self-association may provide an amplification mechanism to promote the activation of RIPK1 kinase activity for mediating signal transduction to lead to cell death. Our results also suggest that the activation of RIPK1 may be regulated by its concentration as increased expression of RIPK1 under pathological conditions may promote its dimerization and activation. RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinase-dependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis.

Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

Significance This study demonstrates a distinct mode of RIPK1 activation mediated by a detergent-insoluble, highly ubiquitinated, activated RIPK1 species (iuRIPK1) which functions as a critical intermediate between TNF-receptor-associated complex I and assembly of the cytosolic caspase activation platform complex II in RIPK1-dependent apoptosis (RDA). By conducting a systematic screen for RDA regulators, we reveal the regulation of iuRIPK1 by Parkinson’s disease (PD)-associated LRRK2, E3 ubiquitin ligase c-Cbl, and ALS-associated NEK1. These results point to possible mechanistic links between RIPK1-mediated apoptosis and neurodegenerative diseases such as ALS and PD. Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)—the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed “iuRIPK1.” iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson’s disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.