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981.
982.
THO2, a core member of the THO/TREX complex,is required for microRNA production in Arabidopsis 下载免费PDF全文
Anchilie G. Francisco‐Mangilet Patricia Karlsson Myung‐Hee Kim Hyeon Ju Eo Sung Aeong Oh Jeong Hoe Kim Franceli Rodrigues Kulcheski Soon Ki Park Pablo Andrés Manavella 《The Plant journal : for cell and molecular biology》2015,82(6):1018-1029
The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA‐dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA‐targeted mRNAs. Yeast two‐hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA‐processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine‐rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing. 相似文献
983.
Autophagy is an important catabolic program to respond to a variety of cellular stresses by forming a double membrane vesicle, autophagosome. Autophagy plays key roles in various cellular functions. Accordingly, dysregulation of autophagy is closely associated with diseases such as diabetes, neurodegenerative diseases, cardiomyopathy, and cancer. In this sense, autophagy is emerging as an important therapeutic target for disease control. Among the autophagy machineries, PIK3C3/VPS34 complex functions as an autophagy-triggering kinase to recruit the subsequent autophagy protein machineries on the phagophore membrane. Accumulating evidence showing that inhibition of PIK3C3/VPS34 complex successfully inhibits autophagy makes the complex an attractive target for developing autophagy inhibitors. However, one concern about PIK3C3/VPS34 complex is that many different PIK3C3/VPS34 complexes have distinct cellular functions. In this study, we have developed an in vitro PIK3C3/VPS34 complex monitoring assay for autophagy inhibitor screening in a high-throughput assay format instead of targeting the catalytic activity of the PIK3C3/VPS34 complex, which shuts down all PIK3C3/VPS34 complexes. We performed in vitro reconstitution of an essential autophagy-promoting PIK3C3/VPS34 complex, Vps34–Beclin1–ATG14L complex, in a microwell plate (96-well format) and successfully monitored the complex formation in many different conditions. This PIK3C3/VPS34 complex protein assay would provide a reliable tool for the screening of autophagy-specific inhibitors. 相似文献
984.
Jeong Jin Ahn Ji-Hoon Kim Youngjoo Kim Ji Young Hong Gi Won Kim Seung Yong Hwang 《Analytical biochemistry》2015
Locked nucleic acid (LNA) is a modified RNA nucleotide that can be incorporated at specific positions to generate probes with the desired length, melting temperature (TM), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the TM of a single dual-labeled LNA probe. Using this method, two varieties of the hairtail fish Trichiurus lepturus can be distinguished from each other, as well as from Trichiurus japonicus, based on a 1- to 2-bp difference in a fragment of mitochondrial cytochrome oxidase subunit 1. The shift in TM was 15 °C for a 1-bp mismatch and 27 °C for a 2-bp mismatch, indicating remarkable specificity. We anticipate that the method will be widely useful in applications such as species identification that require accurate, multiplex, and efficient detection of DNA polymorphisms. 相似文献
985.
Biomimetic repeat protein derived from Xenopus tropicalis for fibrous scaffold fabrication 下载免费PDF全文
Yunkyeoung Kwon Yun Jung Yang Dooyup Jung Byeong Hee Hwang Hyung Joon Cha 《Biopolymers》2015,103(12):659-664
Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiber‐based sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein‐based material. The codon‐optimized recombinant frog repeat protein, referred to as ‘xetro’, was produced at a high rate in a bacterial system, and an acid extraction‐based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet‐spun xetro microfibers demonstrated about 2‐ and 1.5‐fold higher tensile strength compared with synthetic polymer polylactic acid and cross‐linked collagen, respectively. In addition, the wet‐spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber‐based material for biomedical engineering. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 659–664, 2015. 相似文献
986.
Naoyuki Kondo Mariana Marin Jeong Hwa Kim Tanay M. Desai Gregory B. Melikyan 《The Journal of biological chemistry》2015,290(10):6558-6573
Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. 相似文献
987.
Hyojin Park Sungwoon Lee Pravesh Shrestha Jihye Kim Jeong Ae Park Yeongrim Ko Young Ho Ban Dae-Young Park Sang-Jun Ha Gou Young Koh Victor Sukbong Hong Naoki Mochizuki Young-Myeong Kim Weontae Lee Young-Guen Kwon 《The Journal of cell biology》2015,211(3):619-637
The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor. 相似文献
988.
Youngsil Choi Mi-Jung Kwon Yangmi Lim Ji-Hye Yun Weontae Lee Eok-Soo Oh 《The Journal of biological chemistry》2015,290(27):16943-16953
Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed intermolecular interactions in vitro, and the GXXXG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans. 相似文献
989.
Wijeong Jang Ji Young Kim Shanyu Cui Juyeon Jo Byoung-Cheol Lee Yeonwoo Lee Ki-Sun Kwon Chul-Seung Park Changsoo Kim 《The Journal of biological chemistry》2015,290(4):2521-2528
Calcium-permeable and thermosensitive transient receptor potential (TRP) channels mediate the nociceptive transduction of noxious temperature in Drosophila nociceptors. However, the underlying molecular mechanisms are not completely understood. Here we find that Subdued, a calcium-activated chloride channel of the Drosophila anoctamin family, functions in conjunction with the thermo-TRPs in thermal nociception. Genetic analysis with deletion and the RNAi-mediated reduction of subdued show that subdued is required for thermal nociception in nociceptors. Further genetic analysis of subdued mutant and thermo-TRP mutants show that they interact functionally in thermal nociception. We find that Subdued expressed in heterologous cells mediates a strong chloride conductance in the presence of both heat and calcium ions. Therefore, our analysis suggests that Subdued channels may amplify the nociceptive neuronal firing that is initiated by thermo-TRP channels in response to thermal stimuli. 相似文献
990.
Nami Kim Jung Ok Lee Hye Jeong Lee Hyung Ip Kim Joong Kwan Kim Yong Woo Lee Soo Kyung Lee Su Jin Kim Sun Hwa Park Hyeon Soo Kim 《The Journal of biological chemistry》2015,290(33):20438-20447
Docosahexaenoic acid (DHA) is an endogenous ligand of G protein-coupled receptor 120 (GPR120). However, the mechanisms underlying DHA action are poorly understood. In this study, DHA stimulated glucose uptake in the skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner. GPR120-mediated increase in intracellular Ca2+ was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca2+/calmodulin-dependent protein kinase kinase blocked DHA-induced glucose uptake. DHA and GW9508, a GPR120 agonist, increased GPR120 expression. DHA-mediated glucose uptake was not observed in GPR120 knockdown conditions. DHA increased AMPK phosphorylation, glucose uptake, and intracellular Ca2+ concentration in primary cultured myoblasts. Taken together, these results indicated that the beneficial metabolic role of DHA was attributed to its ability to regulate glucose via the GPR120-mediated AMPK pathway in the skeletal muscles. 相似文献