作者机构：
[Yan, Lin; Zhao, Jingyan; Peng, Jianxin; Liu, Kaiyu; Yan, Xiumei; Yang, Yongbo; Ma, Haihao; Song, Jiping; Peng, Rong] Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Peoples R China.
通讯机构：
[Peng, JX; Liu, KY] C;Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Peoples R China.
关键词：
Apaf-1;apoptosis;caspase;Spodoptera litura
摘要：
Simple Summary Apoptosis plays an important role in both the development of lepidopteran insects and the elimination of cells. The apoptosis signal pathways are well documented in mammals and Drosophila melanogaster. However, it remains less clear in lepidopteran insects. This study characterized the apoptotic protease activating factor-1 (Apaf-1) from Spodoptera litura. The results showed that S. litura Apaf-1 (Sl-Apaf-1) is similar to the mammalian Apaf-1. Sl-Apaf-1 consists of a caspase recruitment domain (CARD), as well as nucleotide-binding and oligomerization domain (NOD) and the C-terminal WD40-repeat domain (WD), and interacts with Sl-caspase-5 (a homologue of mammalian caspase-9). The activated Sl-caspase-5 can cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly causes apoptosis. The apoptosis signal pathway is conserved between lepidopteran insects and mammals. Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 x 10(6) M-1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.
作者机构：
[Hai-Hao Ma; Yue-Min Ma; Kai-Yu Liu; Jian-Xin Peng; Hua-Zhu Hong; Rong Peng] School of Life Science,Central China Normal University
会议名称：
The 5th International Symposium on Insect Physiology, Biochemistry and Molecular Biology
会议时间：
2015-06-15
会议地点：
中国广东广州
摘要：
Sterol carrier protein-2(SCP-2), a nonspecific intracellular lipid transfer protein, plays an important role in the growth and development of insects. It has been shown that SCP-2 is involved in the c
摘要：
Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelium air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR, and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (X. Deng, Z. Yan, F. Cheng, J. F. Engelhardt, and J. Qiu, PLoS Pathog, 12:e1005399, 2016, https://doi.org/10.1371/journal.ppat.1005399), and HBoV1 replicates only in terminally differentiated, nondividing cells. This report builds on the previous discovery that the replication of HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral nonstructural protein NS1 is sufficient to induce the DDR and the activation of the three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both the replication of HBoV1 DNA and the downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA replication factors and major DNA repair DNA polymerases (polymerase eta [Pol eta] and polymerase kappa [Pol kappa]) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of the DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest. IMPORTANCE The parvovirus human bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support the replication of the duplex genome of this virus and allow the production of progeny virions. In this study, we demonstrate that HBoV1 induces a DDR that plays significant roles in the replication of the viral DNA and the production of progeny virions in HEK293 cells. We also show that both cellular DNA replication factors and DNA repair DNA polymerases colocalize within centers of viral DNA replication and that Pol eta and Pol kappa play an important role in HBoV1 DNA replication. Whereas the DDR that leads to the replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 has been shown to be able to activate all three PI3KKs (ATM, ATR, and DNA-PKcs).
作者机构：
[Xu, Peng; Liu, Kaiyu; Peng, Jianxin] Cent China Normal Univ, Sch Life Sci, Wuhan, Peoples R China.;[Xu, Peng; Qiu, Jianming; Zou, Wei; Deng, Xuefeng; Ganaie, Safder S.] Univ Kansas, Med Ctr, Dept Microbiol Mol Genet & Immunol, Kansas City, KS 66103 USA.;[Zhou, Zhe; Wang, Shengqi] Beijing Inst Radiat Med, Dept Biotechnol, Beijing, Peoples R China.;[Ye, Shui Qing; Xiong, Min] Childrens Mercy Hosp, Dept Pediat, Kansas City, MO 64108 USA.;[Ye, Shui Qing; Xiong, Min] Childrens Mercy Hosp, Dept Biomed & Hlth Informat, Kansas City, MO 64108 USA.
通讯机构：
[Liu, Kaiyu] C;[Qiu, Jianming] U;[Wang, Shengqi] B;Cent China Normal Univ, Sch Life Sci, Wuhan, Peoples R China.;Univ Kansas, Med Ctr, Dept Microbiol Mol Genet & Immunol, Kansas City, KS 66103 USA.
关键词：
Phosphorylation;Cell cycle and cell division;Cyclins;Flow cytometry;DNA replication;Gene expression;Synthesis phase;Transactivation
摘要：
Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (?100 W/cm2 ) live-cell SR imaging at ?60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.
作者机构：
[Liu, Kaiyu; Peng, Jianxin; Ma, Haihao; Peng, Rong; Ma, Yuemin] Cent China Normal Univ, Sch Life Sci, Wuhan, Peoples R China.;[Liu, Xuehui; Xu, Pingyong] Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100080, Peoples R China.;[Lan, Que; Dyer, David H.] Univ Wisconsin, Coll Agr & Life Sci, Dept Biochem & Entomol, Madison, WI 53706 USA.;[Hong, Huazhu] Wuhan Inst Bioengn, Sch Life Sci, Wuhan, Peoples R China.
通讯机构：
[Peng, Jianxin] C;Cent China Normal Univ, Sch Life Sci, Wuhan, Peoples R China.
摘要：
The cotton bollworm, Helicoverpa armigera, has developed strong resistance to many insecticides. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new target. In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. HaSCP-2 composed of five α-helices and four stranded β-sheets. The folds of α-helices and β-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. Several sterols and fatty acids could interact with HaSCP-2 via important hydrophobic sites, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51, F53, F89, F110, I117 and Q131 may be the key functional sites. HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo. Our study describes for the first time a NMR structure of SCP-2 in lepidopteran H. armigera and reveals its important function in cholesterol uptake, which facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.
摘要：
Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.
摘要：
The presence and abundance of proteins in the plasma membrane (PM) is regulated by exocytosis and endocytosis. Exocytosis is required for the maintenance of cellular homeostasis following stimulation, whereas endocytosis is important for the reuse and degradation of PM and receptor proteins. Through the specific labeling of proteins on the PM, the process of endocytosis can be tracked and monitored in living cells. Several genetic tags and site-specific labeling approaches that involve the coupling of small organic molecules to tag-fused proteins through either self-labeling or enzymatic ligation have been developed for this purpose (Chen et al., 2005; George et al., 2004; Hoffmann et al., 2010; Keppler et al., 2003; Los et al., 2008; Uttamapinant et al., 2010). However, these methods require a long time for an efficient labeling.
