Analysis of conformational motions and related key residue interactions responsible for a specific function of proteins with elastic network model

Protein collective motions play a critical role in many biochemical processes. How to predict the functional motions and the related key residue interactions in proteins is important for our understanding in the mechanism of the biochemical processes. Normal mode analysis (NMA) of the elastic network model (ENM) is one of the effective approaches to investigate the structure-encoded motions in proteins. However, the motion modes revealed by the conventional NMA approach do not necessarily correspond to a specific function of protein. In the present work, a new analysis method was proposed to identify the motion modes responsible for a specific function of proteins and then predict the key residue interactions involved in the functional motions by using a perturbation approach. In our method, an internal coordinate that accounts for the specific function was introduced, and the Cartesian coordinate space was transformed into the internal/Cartesian space by using linear approximation, where the introduced internal coordinate serves as one of the axes of the coordinate space. NMA of ENM in this internal/Cartesian space was performed and the function-relevant motion modes were identified according to their contributions to the specific function of proteins. Then the key residue interactions important for the functional motions of the protein were predicted as the interactions whose perturbation largely influences the fluctuation along the internal coordinate. Using our proposed methods, the maltose transporter (MalFGK₂) from E. Coli was studied. The functional motions and the key residue interactions that are related to the channel-gating function of this protein were successfully identified.     

4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

转基因大豆MON89788芯片式数字PCR定量方法的建立

针对抗虫耐除草剂大豆转基因品系MON89788,从转基因植物基因组DNA的提取、核酸模板的质量和浓度控制、引物探针的筛选、PCR反应过程的建立等方面建立了一套完整的转基因大豆芯片式dPCR定量检测方法。本实验也对该方法的重复性和定量检测限进行考察。10组5%转基因品系大豆MON89788样品定量重复性RSD在1.17%-9.97%之间,均满足国际上转基因定量结果RSD小于25%的要求。用该方法对转基因含量为5%、1%、0.1%的大豆MON89788进行定量检测,其定量结果为5.20%、0.94%和0.11%,RSD分别为6.2%、3.6%和15.2%。该检测方法的定量限达到0.1%,能满足欧盟对转基因定量标识0.9%的要求。将本实验建立的方法用于转基因大豆的定量检测,能为规范我国转基因监管工作的实施提供强有力的技术支撑。     

Cucurbitacin I Induces Protective Autophagy in Glioblastoma in Vitro and in Vivo

There is an urgent need for new therapeutic avenues to improve the outcome of patients with glioblastoma multiforme (GBM). Current studies have suggested that cucurbitacin I, a natural selective inhibitor of JAK2/STAT3, has a potent anticancer effect on a variety of cancer cell types. This study showed that autophagy and apoptosis were induced by cucurbitacin I. Exposure of GBM cells to cucurbitacin I resulted in pronounced apoptotic cell death through activating bcl-2 family proteins. Cells treatment with cucurbitacin I up-regulated Beclin 1 and triggered autophagosome formation and accumulation as well as conversion of LC3I to LC3II. Activation of the AMP-activated protein kinase/mammalian target of rapamycin/p70S6K pathway, but not the PI3K/AKT pathway, occurred in autophagy induced by cucurbitacin I, which was accompanied by decreased hypoxia-inducible factor 1α. Stable overexpression of hypoxia-inducible factor 1α induced by FG-4497 prevented cucurbitacin I-induced autophagy and down-regulation of bcl-2. Knockdown of beclin 1 or treatment with the autophagy inhibitor 3-methyladenine also inhibited autophagy induced by cucurbitacin I. A coimmunoprecipitation assay showed that the interaction of Bcl-2 and Beclin 1/hVps34 decreased markedly in cells treated with cucurbitacin I. Furthermore, knockdown of beclin 1 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cucurbitacin I-induced apoptosis. Finally, a xenograft model provided additional evidence for the occurrence of cucurbitacin I-induced apoptosis and autophagy in vitro. Our findings provide new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cell death and may provide an efficacious therapy for patients harboring GBM.     

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

Heterogeneity of Regional Brain Atrophy Patterns Associated with Distinct Progression Rates in Alzheimer’s Disease

We aimed to identify and characterize subtypes of Alzheimer’s disease (AD) exhibiting different patterns of regional brain atrophy on MRI using age- and gender-specific norms of regional brain volumes. AD subjects included in the Alzheimer''s Disease Neuroimaging Initiative study were classified into subtypes based on standardized values (Z-scores) of hippocampal and regional cortical volumes on MRI with reference to age- and gender-specific norms obtained from 222 cognitively normal (CN) subjects. Baseline and longitudinal changes of clinical characteristics over 2 years were compared across subtypes. Whole-brain-level gray matter (GM) atrophy pattern using voxel-based morphometry (VBM) and cerebrospinal fluid (CSF) biomarkers of the subtypes were also investigated. Of 163 AD subjects, 58.9% were classified as the “both impaired” subtype with the typical hippocampal and cortical atrophy pattern, whereas 41.1% were classified as the subtypes with atypical atrophy patterns: “hippocampal atrophy only” (19.0%), “cortical atrophy only” (11.7%), and “both spared” (10.4%). Voxel-based morphometric analysis demonstrated whole-brain-level differences in overall GM atrophy across the subtypes. These subtypes showed different progression rates over 2 years; and all subtypes had significantly lower CSF amyloid-β_1–42 levels compared to CN. In conclusion, we identified four AD subtypes exhibiting heterogeneous atrophy patterns on MRI with different progression rates after controlling the effects of aging and gender on atrophy with normative information. CSF biomarker analysis suggests the presence of Aβ neuropathology irrespective of subtypes. Such heterogeneity of MRI-based neuronal injury biomarker and related heterogeneous progression patterns should be considered in clinical trials and practice with AD patients.     

Evaluation of Mercury Contamination in Fungi Boletus Species from Latosols,Lateritic Red Earths,and Red and Yellow Earths in the Circum-Pacific Mercuriferous Belt of Southwestern China

For the first time, highly elevated levels of mercury (Hg) have been documented for several species of the edible Fungi genus Boletus growing in latosols, lateritic red earths, and red and yellow earths from the Yunnan province of China. Analysis of Hg concentrations in the genus suggests that geogenic Hg is the dominant source of Hg in the fungi, whereas anthropogenic sources accumulate largely in the organic layer of the forest soil horizon. Among the 21 species studied from 32 locations across Yunnan and 2 places in Sichuan Province, the Hg was found at elevated level in all samples from Yunnan but not in the samples from Sichuan, which is located outside the mercuriferous belt. Particularly abundant in Hg were the caps of fruiting bodies of Boletus aereus (up to 13 mg kg^-1 dry matter), Boletus bicolor (up to 5.5 mg kg^-1 dry matter), Boletus edulis (up to 22 mg kg^-1 dry matter), Boletus luridus (up to 11 mg kg^-1 dry matter), Boletus magnificus (up to 13 mg kg^-1 dry matter), Boletus obscureumbrinus (up to 9.4 mg kg^-1 dry matter), Boletus purpureus (up to 16 mg kg^-1 dry matter), Boletus sinicus (up to 6.8 mg kg^-1 dry matter), Boletus speciosus (up to 4.9mg kg^-1 dry matter), Boletus tomentipes (up to 13 mg kg^-1 dry matter), and Boletus umbriniporus (up to 4.9 mg kg^-1 dry matter). Soil samples of the 0–10 cm topsoil layer from the widely distributed locations had mercury levels ranging between 0.034 to 3.4 mg kg^-1 dry matter. In Yunnan, both the soil parent rock and fruiting bodies of Boletus spp. were enriched in Hg, whereas the same species from Sichuan, located outside the mercuriferous belt, had low Hg concentrations, suggesting that the Hg in the Yunnan samples is mainly from geogenic sources rather than anthropogenic sources. However, the contribution of anthropogenically-derived Hg sequestered within soils of Yunnan has not been quantified, so more future research is required. Our results suggest that high rates of consumption of Boletus spp. from Yunnan can deliver relatively high doses of Hg to consumers, but that rates can differ widely because of large variability in mercury concentrations between species and locations.