Structure and Functional Implications of the Human Rad9-Hus1-Rad1 Cell Cycle Checkpoint Complex

Cellular DNA lesions are efficiently countered by DNA repair in conjunction with delays in cell cycle progression. Previous studies have demonstrated that Rad9, Hus1, and Rad1 can form a heterotrimeric complex (the 9-1-1 complex) that plays dual roles in cell cycle checkpoint activation and DNA repair in eukaryotic cells. Although the 9-1-1 complex has been proposed to form a toroidal structure similar to proliferating cell nuclear antigen (PCNA), which plays essential roles in DNA replication and repair, the structural basis by which it performs different functions has not been elucidated. Here we report the crystal structure of the human 9-1-1 complex at 3.2 Å resolution. The crystal structure, together with biochemical assays, reveals that the interdomain connecting loops (IDC loop) of hRad9, hHus1, and hRad1 are largely divergent, and further cocrystallization study indicates that a PCNA-interacting box (PIP box)-containing peptide derived from hFen1 binds tightly to the interdomain connecting loop of hRad1, providing the molecular basis for the damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA reveals other unique structural features of the 9-1-1 complex that are proposed to contribute to DNA damage recognition.Cellular DNA damage triggers the activation of the cell cycle checkpoint, leading to a delay or arrest in cell cycle progression to prevent replication and inducing DNA damage repair (1, 2). In response to DNA damage, the 9-1-1³ complex can be loaded onto DNA lesion sites by Rad17-RFC2–5 (which consists of one large subunit, Rad17, and four small subunits, RFC2–5), where it triggers the activation of the cell cycle checkpoint (3, 4). Moreover, the 9-1-1 complex can also directly participate in DNA repair via physical association with many factors involved in base excision repair (BER), translesion synthesis, homologous recombination, and mismatch repair pathways (5–9).Although both the 9-1-1 and the PCNA complexes perform critical functions in eukaryotic cells with predicted similar structures (10), their specific roles are distinct. First, the 9-1-1 complex is a DNA damage sensor in the cell cycle checkpoint but does not function as a scaffold for the major DNA replication factors; however, PCNA plays exactly the opposite role (1, 11). Second, although both the complexes function in DNA repair, their specific activities are different. Previous observations indicated that some BER enzymes, such as MYH (MutY glycosylate homolog) (12), TDG (thymine DNA glycosylate) (7), and NEIL (Nei-like glycosylate) (8), interact with the 9-1-1 complex via motifs that are located outside the conserved PCNA-interacting box (the PIP box), implying that the 9-1-1 complex functions as a damage repair-specific clamp, in contrast to PCNA. However, the structural basis for this hypothesis remains unclear. Another important unresolved issue concerns the damage-sensing mechanism of the 9-1-1 complex. During the DNA replication process, the PCNA·RFC clamp·clamp loader specifically recognizes the primer-template junction (13). However, the molecular basis by which the 9-1-1·Rad17-RFC2–5 clamp·clamp loader specifically recognizes the damaged DNA is little known. To address these questions, we performed structural and biochemical studies on the 9-1-1 complex.     

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.     

RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis

Acetylcholinesterase （ACHE） expression may be induced during apoptosis in various cell types. Here, we used the C-terminal of AChE to screen the human fetal brain library and found that it interacted with Ran-binding protein in the microtubule-organizing center （RanBPM）. This interaction was further confirmed by coimmunoprecipitation analysis. In HEK293T cells, RanBPM and AChE were heterogeneously expressed in the cisplatin-untreated cytoplasmic extracts and in the cisplatin-treated cytoplasmic or nuclear extracts. Our previous studies performed using morphologic methods have shown that AChE translocates from the cytoplasm to the nucleus during apoptosis. Taken together, these results suggest that RanBPM is an AChE-interacting protein that is translocated from the cytoplasm into the nucleus during apoptosis, similar to the translocation observed in case of ACHE.     

Development of plant communities after restoration of the Antaibao mining site, China

To investigate the dynamic changes in the artificial vegetation in an abandoned mining site, we analyzed the relationships among community types, environmental variables and community structure in the process of vegetation restoration in the Antaibao mining site, China by survey of the communities and use of biological dating methods. By means of the quantitative classification method (two-way indicator-species analysis, TWINSPAN) and the ordination technique (de-trended correspondence analysis, DCA; and de-trended canonical correspondence analysis, DCCA), the plant communities were classified into seven groups: community I, Robinia pseudoacacia + Pinus tabulaeformis-Caragana korshinskii-Agropyron cristatum; community II, Robinia pseudoacacia-Hippophae rhamnoides-Artemisia capillaries; community III, Ulmus pumila-Elaeagnus angustifolia-Artemisia capillaries;community IV, Caragana korshinskii-Agropyron cristatum + Artemisia capillaries; community V, Hippophae rhamnoides-Elymus dahuricus; community VI, Elaeagnus angustifolia + Hippophae rhamnoides-Brassica jucea;community VII, Hippophae rhamnoides + Elaeagnus angustifolia-Salsola collina. We conclude that the community types and diversity are mainly influenced by the succession time and the soil organic matter content. The forest community is more adaptable to the special inhabitation than the shrub community.     

Novel strategies to mine alcoholism-related haplotypes and genes by combining existing knowledge framework

High-throughout single nucleotide polymorphism detection technology and the existing knowledge provide strong support for mining the disease-related haplotypes and genes. In this study, first, we apply four kinds of haplotype identification methods (Confidence Intervals, Four Gamete Tests, Solid Spine of LD and fusing method of haplotype block) into high-throughout SNP genotype data to identify blocks, then use cluster analysis to verify the effectiveness of the four methods, and select the alcoholism-related SNP haplotypes through risk analysis. Second, we establish a mapping from haplotypes to alcoholism-related genes. Third, we inquire NCBI SNP and gene databases to locate the blocks and identify the candidate genes. In the end, we make gene function annotation by KEGG, Biocarta, and GO database. We find 159 haplotype blocks, which relate to the alcoholism most possibly on chromosome 1∼22, including 227 haplotypes, of which 102 SNP haplotypes may increase the risk of alcoholism. We get 121 alcoholism-related genes and verify their reliability by the functional annotation of biology. In a word, we not only can handle the SNP data easily, but also can locate the disease-related genes precisely by combining our novel strategies of mining alcoholism-related haplotypes and genes with existing knowledge framework. Supported by the National Natural Science Foundation of China (Grant Nos. 30570424, 60601010 and 30600367), the National High-Tech Research and Development Program of China, (Grant No.2007AA02Z329), the Key Science and Technology Program of Heilongjiang Province(Grant No.GB03C602-4), Natural Science Foundation of Heilongjiang Province (Grant No. F2008-02), Youth Science Foundation of Harbin Medical University (Grant No. 060045) and Science Foundation of Heilongjiang Province Education Department (Grant Nos. 11531113 and 1152hq28).     

In vitro drug release behavior from a novel thermosensitive composite hydrogel based on Pluronic f127 and poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) copolymer

Background  

Most conventional methods for delivering chemotherapeutic agents fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. Novel controlled drug delivery systems are designed to deliver drugs at predetermined rates for predefined periods at the target organ and overcome the shortcomings of conventional drug formulations therefore could diminish the side effects and improve the life quality of the patients. Thus, a suitable controlled drug delivery system is extremely important for chemotherapy.     

Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

Background  

Trehalose synthase (TreS) which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production. This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate. Therefore, new TreS producing bacteria with suitable enzyme properties are expected to be isolated from extreme environment.     

Transgenic Expression of Walleye Dermal Sarcoma Virus rv-cyclin Gene in Zebrafish and Its Suppressive Effect on Liver Tumor Development After Carcinogen Treatment

A retrovirus homologue gene of cellular cyclin D ₁ , walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin–). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin– fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.