The industrial artificial fixation of atmospheric N2 to NH3 is carried out using the Haber–Bosch process that is not only energy‐intensive but emits large amounts of greenhouse gas. Electrochemical reduction offers an environmentally benign and sustainable alternative for NH3 synthesis. Although Mo‐dependent nitrogenases and molecular complexes effectively catalyze the N2 fixation at ambient conditions, the development of a Mo‐based nanocatalyst for highly performance electrochemical N2 fixation still remains a key challenge. Here, greatly boosted electrocatalytic N2 reduction to NH3 with excellent selectivity by defect‐rich MoS2 nanoflowers is reported. In 0.1 m Na2SO4, this catalyst attains a high Faradic efficiency of 8.34% and a high NH3 yield of 29.28 µg h−1 mg−1cat. at −0.40 V versus reversible hydrogen electrode, much larger than those of defect‐free counterpart (2.18% and 13.41 µg h−1 mg−1cat.), with strong electrochemical stability. Density functional theory calculations show that the potential determining step has a lower energy barrier (0.60 eV) for defect‐rich catalyst than that of defect‐free one (0.68 eV).

DNA replication initiation is tightly controlled so that each origin only fires once per cell cycle. Cell cycle-dependent Cdt1 degradation plays an essential role in DNA replication control, as overexpression of Cdt1 leads to re-replication. In this study, we investigated the mechanisms of Cdt1 degradation in mammalian cells. We showed that the F-box protein Skp2 specifically interacted with human Cdt1 in a phosphorylation-dependent manner. The SCF Skp2 complex ubiquitinated Cdt1 both in vivo and in vitro. Down-regulation of Skp2 or disruption of the interaction between Cdt1 and Skp2 resulted in a stabilization and accumulation of Cdt1. These results suggest that the SCF Skp2 -mediated ubiquitination pathway may play an important role in the cell cycle-dependent Cdt1 degradation in mammalian cells.In all eukaryotic cells, DNA replication is tightly controlled to ensure that initiation of replication occurs only once per cell cycle (1-3). The key to this regulation is the formation of prereplication complexes (pre-RCs) 1 by loading MCM proteins to replication origins (4, 5). Studies from yeast and Xenopus showed that Cdc6 (termed as Cdc18 in Schizosaccharomyces pombe) and Cdt1 are required for MCM proteins to associate with chromatin (6 -14). In fission yeast, the protein levels of both Cdc18 and Cdt1 are tightly controlled during the cell cycle and Cdc18 and Cdt1 only appear in G 1 , which is important to prevent re-formation of pre-RCs after DNA is replicated in S phase (15)(16)(17)(18). Cdc18 is targeted for ubiquitination-mediated degradation when cells enter S phase of the cell cycle (17).In mammalian cells, Cdc6 protein levels remain almost constant throughout G 1 , S, and G 2 phases (19 -24). After the onset of S phase, Cdc6 is phosphorylated and exported to the cytoplasm (20,23,25,26). In contrast to Cdc6, the protein level of Cdt1 fluctuates during the cell cycle (27). It accumulates in early G 1 phase of the cell cycle when replication is licensed and disappears at the onset of S phase. The correlation of Cdt1 accumulation and assembly of pre-RCs in early G 1 phase suggests that Cdt1 may play an important role in the control of replication licensing in mammalian cells. This idea is further supported by the observation that overexpression of Cdt1 promotes DNA re-replication in p53 deficient cells (28).It has been shown that in mammalian cells, while Cdt1 protein level varies during the cell cycle, the mRNA level of Cdt1 remains relatively constant at different cell cycle stages (27). In the presence of proteasome inhibitors, Cdt1 accumulates in S phase when it is normally absent. This suggests that proteasome-dependent degradation may regulate the Cdt1 level during the cell cycle in mammalian cells. However, the detailed mechanisms of Cdt1 degradation remain unknown.The ubiquitin-dependent proteolytic pathway plays an important role for protein degradation (29). The conjugation of polyubiquitin chains to substrates requires enzymes, E1 (a ubiquitin-activating enzyme), E2 (a ubiquitin-conjugating enzyme...

Phosphorylation of Thr-68 by the ataxia telangiectasia-mutated is necessary for efficient activation of Chk2 when cells are exposed to ionizing radiation. By an unknown mechanism, this initial event promotes additional autophosphorylation events including modifications of Thr-383 and Thr-387, two amino acid residues located within the activation loop segment within the Chk2 catalytic domain. Chk2 and related kinases possess one or more Forkhead-associated (FHA) domains that are phosphopeptide-binding modules believed to be crucial for their checkpoint control activities. We show that the Chk2 FHA domain is dispensable for Thr-68 phosphorylation but necessary for efficient autophosphorylation in response to ionizing radiation. Phosphorylation of Thr-68 promotes oligomerization of Chk2 by serving as a specific ligand for the FHA domain of another Chk2 molecule. In addition, Chk2 phosphorylates its own FHA domain, and this modification reduces its affinity for Thr-68-phosphorylated Chk2. Thus, activation of Chk2 in irradiated cells may occur through oligomerization of Chk2 via binding of the Thr-68-phosphorylated region of one Chk2 to the FHA domain of another. Oligomerization of Chk2 may therefore increase the efficiency of trans-autophosphorylation resulting in the release of active Chk2 monomers that proceed to enforce checkpoint control in irradiated cells.The maintenance of genomic integrity following DNA damage requires the coordinated actions of DNA repair and cell cycle checkpoint control. The Chk2/hCds1 protein kinase is activated by DNA damage and phosphorylates several known modulators of cell cycle control including the tumor suppressor proteins, p53 and BRCA1, and Cdc25A phosphatase (1-9). Mutations in the CHK2 gene have been identified in human hereditary and sporadic cancers suggesting that post-translational modifications mediated by Chk2 play important roles in altering the activities of these checkpoint control proteins (10 -14). Therefore, exploring the mechanisms by which Chk2 activity is regulated will enhance our understanding of the complex network of signaling pathways that serve to limit tumor development.Chk2 is a direct target and major effector of the ATM 1 kinase, a key regulator of cell cycle checkpoint control in irradiated cells (for review see Ref. 15). In response to ionizing radiation (IR) and double-stranded DNA breaks, ATM phosphorylates Chk2 on Thr-68 within the amino-terminal SQ/TQrich domain, and this event is necessary for efficient activation of the Chk2 kinase (16 -18). Recent evidence suggests that Chk2 is phosphorylated on Thr-383 and Thr-387 in a phosphothreonine 68 (Thr(P)-68)-dependent manner (19). These residues are located within the activation loop segment of Chk2 and therefore may be Chk2 autophosphorylation sites that increase the specific activity of the enzyme when modified (19,20). This model is consistent with our observations that a catalytic inactive Chk2 fails to exhibit phosphorylationdependent mobility shifts upon SDS-PAGE even though clearly phosphoryla...

SUMMARY Hypoxic stress and hypoxia-inducible factors (HIFs) play important roles in a wide range of tumors. We demonstrate that SPOP, which encodes an E3 ubiquitin ligase component, is a direct transcriptional target of HIFs in clear cell renal cell carcinoma (ccRCC). Furthermore, hypoxia results in cytoplasmic accumulation of SPOP which is sufficient to induce tumorigenesis. This tumorigenic activity occurs through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis, including the tumor suppressor PTEN, ERK phosphatases, the pro-apoptotic molecule Daxx and the Hedgehog pathway transcription factor Gli2. Knockdown of SPOP specifically kills ccRCC cells, indicating that it may be a promising therapeutic target. Collectively, our results indicate that SPOP serves as a regulatory hub to promote ccRCC tumorigenesis.