Structural and functional variations in human apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.     

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells

MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.     

An ion-channel forming protein produced by Entamoeba histolytica.

We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery. A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction. Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes. Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude. The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV. In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease. Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g. It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS. Under these conditions it has an apparent mol. wt. of 13 000 daltons.     

Fractal model of ion-channel kinetics

Markov models with discrete states, such as closed in equilibrium with closed in equilibrium with open have been widely used to model the kinetics of ion channels in the cell membrane. In these models the transition probabilities per unit time (the kinetic rate constants) are independent of the time scale on which they are measured. However, in many physical systems, a property, L, depends on the scale, epsilon, at which it is measured such that L(epsilon) alpha epsilon 1-D where D is the fractal dimension. Such systems are said to be 'fractal'. Based on the assumption that the kinetic rates are given by k(t) alpha t1-D we derive a fractal model of ion-channel kinetics. This fractal model has fewer adjustable parameters, is more consistent with the dynamics of protein conformations, and fits the single-channel recordings from the corneal endothelium better than the discrete-state Markov model.     

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting

Background

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3’s action remain unanswered.

Results

We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants.

Conclusions

Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on mitochondria.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0152-0) contains supplementary material, which is available to authorized users.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID''s competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.     

Protein microarrays as tools for functional proteomics

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.     

利用番茄突变体进行功能基因组学研究

利用番茄突变体进行遗传学研究和育种已有几十年的历史,然而能从分子水平上识别的番茄突变体却为数不多。目前已经获得了大量的番茄序列信息,但仅明确了少数基因的功能。应用饱和突变群体的发展方向,检测突变体和挖掘序列数据的新方法,架起了研究番茄基因和其功能之间的桥梁。     

In vivo study of breast carcinoma radiosensitization by targeting eIF4E

BackgroundEukaryotic initiation factor eIF4E, an important regulator of translation, plays a crucial role in the malignant transformation, progression and radioresistance of many human solid tumors. The overexpression of this gene has been associated with tumor formation in a wide range of human malignancies, including breast cancer. In the present study, we attempted to explore the use of eIF4E as a therapeutic target to enhance radiosensitivity for breast carcinomas in a xenograft BALB/C mice model.Materials and methodsNinety female BALB/C mice transfected with EMT-6 cells were randomly divided into six groups: control, irradiation (IR), pSecX-t4EBP1, pSecX-t4EBP1 + irradiation, pSecX and pSecX + irradiation. At the end of the experiments, all mice were sacrificed, the xenografts were harvested to measure the tumor volume and mass, and the tumor inhibition rates were calculated. Apoptosis was detected with a flow cytometric assay. Immunohistochemistry was used to detect the expression of HIF-1α.ResultsThe xenografts in pSecX-t4EBP1 mice showed a significantly delayed growth and smaller tumor volume, with a higher tumor inhibition rate compared with the control and pSecX groups. A similar result was obtained in the pSecX-t4EBP1 + IR group compared with IR alone and pSecX + irradiation. The expression of HIF-1α in the tumor cells was significantly decreased, while the apoptosis index was much higher.ConclusionspSecX-t4EBP1 can significantly inhibit tumor growth and enhance the radiosensitivity of breast carcinoma xenografts in BALB/C mice. This is possibly associated with the downregulation of HIF-1α expression, which suggests that pSecX-t4EBP1 may serve as an ideal molecular target for the radiosensitization of breast carcinoma.     

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.     

Generation of superoxide anions by leukocytes treated with cytochalasin E.

Guinea pig polymorphonuclear leucocytes reduced cytochrome c when treated with cytochalasin E. The reduction was completely inhibited by superoxide dismutase and manganese ions, which indicates that superoxide anions are generated and released into the outside medium by the treatment. The reduction was inhibited by glycolytic inhibitors and cyclic AMP but not by cyclic GMP. The pattern is similar to the cyanide-insensitive respiration of leucocytes during phagocytosis. Nitroblue tetrazolium was also reduced by the leucocytes treated with the cytochalasin, which was inhibited by manganese ions, glycolytic inhibitors and cyclic AMP but was only partially inhibited by superoxide dismutase.     

基因打靶技术在植物基因功能研究中的应用

水稻、拟南芥等模式植物基因组测序计划的完成,验证预测基因的功能成为植物功能基因组学的重要内容,基因打靶(gene targeting)技术在哺乳动物中的成功应用为该技术在植物上展现了广阔的应用前景。现对植物基因打靶技术的影响因素、基因打靶在植物中的应用现状作一介绍。     

Generation of functional fluorescent BK channels by random insertion of GFP variants

The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the alpha subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The "success rate" of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.