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：
Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in SI-HP cells that are sensitive to CrylAc toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in SI-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced CrylAc-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a CrylAc-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing CrylAc protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the CrylAc-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae. (C) 2017 Elsevier Ltd. All rights reserved.
期刊：
Journal of Virology,2017年91(1):JVI.01831-16 ISSN：0022-538X
通讯作者：
Qiu, Jianming
作者机构：
[Xu, Peng; Shen, Weiran; Qiu, Jianming; Zou, Wei; Deng, Xuefeng] Univ Kansas, Med Ctr, Dept Microbiol Mol Genet & Immunol, Kansas City, KS 66103 USA.;[Xu, Peng; Liu, Kaiyu; Peng, Jianxin] Cent China Normal Univ, Coll Life Sci, Wuhan, Peoples R China.;[Engelhardt, John F.; Yan, Ziying] Univ Iowa, Dept Anat & Cell Biol, Iowa City, IA USA.
通讯机构：
[Qiu, Jianming] U;Univ Kansas, Med Ctr, Dept Microbiol Mol Genet & Immunol, Kansas City, KS 66103 USA.
关键词：
DNA damage;DNA replication;parvovirus
摘要：
<jats:title>ABSTRACT</jats:title>
<jats:p>
Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus
<jats:named-content content-type="genus-species">Bocaparvovirus</jats:named-content>
of the
<jats:named-content content-type="genus-species">Parvoviridae</jats:named-content>
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,
<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://doi.org/10.1371/journal.ppat.1005399">https://doi.org/10.1371/journal.ppat.1005399</jats:ext-link>
), 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 η [Pol η] and polymerase κ [Pol κ]) 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.
</jats:p>
<jats:p>
<jats:bold>IMPORTANCE</jats:bold>
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 η and Pol κ 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).
</jats:p>
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 ( approximately 100 W/cm(2)) live-cell SR imaging at approximately 60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.
期刊：
Insect Biochemistry and Molecular Biology,2015年59:1-17 ISSN：0965-1748
通讯作者：
Liu, Kaiyu
作者机构：
[Liu, Kaiyu; Chen, Zuwen; Peng, Jianxin; Hong, Huazhu; Yang, Yongbo; He, Fei; Ai, Hui; Li, Jianghuai] Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Peoples R China.;[Xiao, Yutao; Liu, Chenxi] Chinese Acad Agr Sci, Inst Plant Protect, State Key Lab Biol Plant Dis & Insect Pests, Beijing 100193, Peoples R China.
通讯机构：
[Liu, Kaiyu] C;Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Peoples R China.
关键词：
ATP-binding cassette transporter;Bacillus thuringiensis;Cadherin;Cytotoxicity;Spodoptera litura cell line
摘要：
Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera aura SI-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the S1ABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SIABCC3 significantly decreased the susceptibility of SI-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SIABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SIABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action. (C) 2015 Elsevier Ltd. All rights reserved.
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：
Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pica value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness. (C) 2014 Elsevier Inc. All rights reserved.
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.
作者机构：
[Gai, Zhongchao; Zhang, Xiaojuan; Liu, Kaiyu; Wang, Xia; Peng, Jianxin; Hong, Huazhu; Li, Yi] Cent China Normal Univ, Coll Life Sci, Wuhan 430079, Hubei Province, Peoples R China.;[Liu, Kaiyu] Cent China Normal Univ, Coll Life Sci, Luoyu Rd 152, Wuhan 430079, Hubei Province, Peoples R China.
通讯机构：
[Liu, Kaiyu] C;Cent China Normal Univ, Coll Life Sci, Luoyu Rd 152, Wuhan 430079, Hubei Province, Peoples R China.
关键词：
Differential proteome;Selection of resistance;Trichoplusia ni;Bacillus thuringiensis;Resistance to Cry1Ac
摘要：
Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.
摘要：
Yeast Atg8 and mammalian microtubule-associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. Sequence analysis reveals that Atg8 proteins are highly conserved in lepidopteran species. The abundance of endogeous Atg8 and the ratios of Atg8 conjugation to phosphatidylethanolamine (Atg8-PE)/Atg8 are different among several lepidopteran cell lines and different tissues of Helicoverpa armigera larvae. Both the density of fluorescent pre-autophagosomal structures with GFP-Ha Atg8 and the abundance of Atg6 are positively correlated with levels of Atg8-PE in different cell lines. The mutant GFP-Atg8(G116A) has lost the function in punctual formation, suggesting that G116 is important for autophagy. Exogenous factors have significant influences on the conversion of Atg8 in lepidopteran cells. Bacillus thuringiensis enhances the degradation of Atg8 in Spodoptera litura Sl-HP cells. Atg8-PE degrades gradually with extension of amino acid starvation, and bafilomycin A1 can block the decrease through the inhibition of autophagosome fusion with lysosome. Interestingly, high pH is more effective than amino acid starvation in Bombyx mori Bme cells to induce the conversion of BmAtg8 to BmAgt8-PE. Change of the quality of fetal bovine serum in the culture medium results in alteration of the ratio of Atg8-PE/Atg8 in some lepidopteran cell lines.
