共查询到20条相似文献,搜索用时 31 毫秒
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Elton Zeqiraj Beatrice Maria Filippi Simon Goldie Iva Navratilova Jér?me Boudeau Maria Deak Dario R. Alessi Daan M. F. van Aalten 《PLoS biology》2009,7(6)
Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations. 相似文献
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The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years. 相似文献
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The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr
protein kinase that is associated with STRAD and MO25 in vivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and
their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning
approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25α can be expressed
in soluble form, whereas the other two, LKB1 and STRADα are found almost exclusively in inclusion bodies. However, using the
tricistronic vector system, functional LKB1-MO25α-STRADα complex was expressed and purified from soluble extracts by sequential
immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion
chromatography, MO25α and STRADα exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex
of 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/mg at approximately
25% purity. As shown by autoradiography, LKB1 and STRADα, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the α-subunit at the Thr-172 site as shown (i) by
Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation
of radioactive phosphate into the α-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK.
Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable
tool for studying its molecular function and regulation. 相似文献
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Marijana Popović Hadžija Marina Korolija Gabrijela Vukadinović Mirko Hadžija 《Central European Journal of Biology》2013,8(6):513-519
Type 1 Diabetes mellitus (T1DM) begins with aberrant inflammatory process followed by auto-destruction in genetically susceptible individuals. Therefore, we hypothesized that gain-of-function allelic variants TNF-α-238A, -308A and PTPN22 1858T could be associated not only with T1DM development but also with the clinical outcome in patients of Bosnia and Herzegovina. A total of 402 subjects were enrolled in the association study. SNPs were determined by PCR-RFLP. Data was analyzed by GraphPad Prism and Sigma Stat 3.5 software. Genotypes frequencies at TNF-α-238 and -308 loci were not statistically different between patients and controls. In contrast, distribution of genotypes at the 1858 position of PTPN22 was significantly different, due to higher frequency of gain-of-function gene variants in patients than controls. Moreover, long term glucose regulation (based on HbA1c level) was significantly worse in patients with the risk TNF-α-308A allele than in patients with non-risk (G) allele. However, patients with the risk allele of both genes (TNF-α-308A and PTPN22 1858T) had the worst glycemic control, suggesting that those two work synergistically. In conclusion, in a cohort from Bosnia and Herzegovina TNF-α-308A allele is significantly associated with the worse long-term glucose control, but PTPN22 1858T allele is significantly associated with diabetes development. 相似文献
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I. D. C. Jansen A. P. Hollander D. J. Buttle V. Everts 《Journal of molecular histology》2010,41(1):9-17
The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage.
Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of
the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen
was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity
of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen.
Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent
without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded
the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable
difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion
of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded
within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the
higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion. 相似文献
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Su K Tian Y Wang J Shi W Luo D Liu J Tong Z Wu J Zhang J Wei L 《DNA and cell biology》2012,31(6):1078-1087
Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α. 相似文献
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Kokoz YM Tsyganova VG Korystova AF Grichenko AS Zenchenko KI Drinyaev VA Mosin VA Kruglyak EB Sterlina TS Victorov AV 《Bioscience reports》1999,19(6):535-546
A natural avermectin complex, aversectin C, was shown to be capable of exerting selective cytostatic and neurotoxic effects on mammalian cells. Specifically, it killed proliferating neuroblastoma B103 cells but was non-toxic for differentiated cells of this culture. The anti-proliferation action of aversectin C was not inhibited by bicuculline or picrotoxin, antagonists of the GABA receptors, and was partly due to the action of avermectin A1, a component of aversectin C. Aversectin C irreversibly suppressed activity of 60% neurons in medial septal slices of the rat brain. More than 55% of them were the GABA- and B1-sensitive neurons whereas the rest, about 45% neurons, were the GABA-insensitive and the neurotoxic effect of aversectin C was caused mainly by the B2 component. 相似文献
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Oluyomi Stephen Adeyemi Yuho Murata Tatsuki Sugi Yongmei Han Kentaro Kato 《Biochemistry and Biophysics Reports》2017
BackgroundToxoplasmosis constitutes a large global burden that is further exacerbated by the shortcomings of available therapeutic options, thus underscoring the urgent need for better anti-Toxoplasma gondii therapy or strategies. Recently, we showed that the anti-parasitic action of inorganic nanoparticles (NPs) could, in part, be due to changes in redox status as well as in the parasite mitochondrial membrane potential.MethodsIn the present study, we explored the in vitro mode of action of the anti-T. gondii effect of NPs by evaluating the contributions of host cellular processes, including the tryptophan pathway and hypoxia-inducing factor activity. NPs, at concentrations ranging from 0.01 to 200 µg/ml were screened for anti-parasitic activity. Sulfadiazine and/or pyrimethamine served as positive controls.ResultsWe found that interplay among multiple host cellular processes, including HIF-1α activity, indoleamine 2,3-dioxygenase activity, and to a larger extent the tryptophan pathway, contribute to the anti-parasitic action of NPs.ConclusionTo our knowledge, this is the first study to demonstrate an effect of NPs on the tryptophan and/or kynurenine pathway.General significanceOur findings deepen our understanding of the mechanism of action of NPs and suggest that modulation of the host nutrient pool may represent a viable approach to the development of new and effective anti-parasitic agents. 相似文献
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Taichi Kakihana Kazutaka Araki Stefano Vavassori Shun-ichiro Iemura Margherita Cortini Claudio Fagioli Tohru Natsume Roberto Sitia Kazuhiro Nagata 《The Journal of biological chemistry》2013,288(41):29586-29594
In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. 相似文献
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Background
EFL (or elongation factor-like) is a member of the translation superfamily of GTPase proteins. It is restricted to eukaryotes, where it is found in a punctate distribution that is almost mutually exclusive with elongation factor-1 alpha (EF-1α). EF-1α is a core translation factor previously thought to be essential in eukaryotes, so its relationship to EFL has prompted the suggestion that EFL has spread by horizontal or lateral gene transfer (HGT or LGT) and replaced EF-1α multiple times. Among green algae, trebouxiophyceans and chlorophyceans have EFL, but the ulvophycean Acetabularia and the sister group to green algae, land plants, have EF-1α. This distribution singles out green algae as a particularly promising group to understand the origin of EFL and the effects of its presence on EF-1α. 相似文献20.
Reactive oxygen species (ROS) play an important role in normal signaling events and excessive ROS are associated with many pathological conditions. The amount of ROS in cells is dependent on both the production of ROS by the mitochondrial electron transport chain and their removal by ROS-detoxifying enzymes. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial functions and a key regulator of the ROS-detoxifying program. However, the impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain is not known. We report here, using mitochondria from muscle creatine kinase–PGC-1α transgenic mice, that PGC-1α does not affect the topology of ROS production, but increases the capacity of complexes I and III to generate ROS. These changes are associated with increased mitochondrial respiration and content of respiratory chain complexes. When normalizing ROS production to mitochondrial respiration, we find that PGC-1α preserves the percentage of free radical leak by the electron transport chain. Together, these data demonstrate that PGC-1α regulates the intrinsic properties of mitochondria in such a way as to preserve a tight coupling between mitochondrial respiration and ROS production. 相似文献