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991.
Donald JE Zhu H Litvinov RI DeGrado WF Bennett JS 《The Journal of biological chemistry》2010,285(49):38658-38665
Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations. 相似文献
992.
Youhei Sohma Qing-xin Hua Ming Liu Nelson B. Phillips Shi-Quan Hu Jonathan Whittaker Linda J. Whittaker Aubree Ng Charles T. Roberts Jr Peter Arvan Stephen B. H. Kent Michael A. Weiss 《The Journal of biological chemistry》2010,285(7):5040-5055
Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis. 相似文献
993.
Jin-Bo Niu Chun-Quan Hua Yuan Liu Guang-Xi Yu Jia-Jia Yang Yin-Ru Li Yan-Bing Zhang Ying-Qiu Qi Jian Song Cheng-Yun Jin Sai-Yang Zhang 《Journal of enzyme inhibition and medicinal chemistry》2021,36(1):1715
Hippo signalling pathway plays a crucial role in tumorigenesis and cancer progression. In this work, we identified an N-aryl sulphonamide-quinazoline derivative, compound 9i as an anti-gastric cancer agent, which exhibited potent antiproliferative ability with IC50 values of 0.36 μM (MGC-803 cells), 0.70 μM (HCT-116 cells), 1.04 μM (PC-3 cells), and 0.81 μM (MCF-7 cells), respectively and inhibited YAP activity by the activation of p-LATS. Compound 9i was effective in suppressing MGC-803 xenograft tumour growth in nude mice without obvious toxicity and significantly down-regulated the expression of YAP in vivo. Compound 9i arrested cells in the G2/M phase, induced intrinsic apoptosis, and inhibited cell colony formation in MGC-803 and SGC-7901 cells. Therefore, compound 9i is to be reported as an anti-gastric cancer agent via activating the Hippo signalling pathway and might help foster a new strategy for the cancer treatment by activating the Hippo signalling pathway regulatory function to inhibit the activity of YAP. 相似文献
994.
Faber JE Szymeczek CL Salvi SS Zhang H 《American journal of physiology. Heart and circulatory physiology》2006,291(5):H2272-H2281
Mechanisms that induce the excessive proliferation of vascular wall cells in hypoxic pulmonary hypertension (PH) are not fully understood. Alveolar hypoxia causes sympathoexcitation, and norepinephrine can stimulate alpha(1)-adrenoceptor (alpha(1)-AR)-dependent hypertrophy/hyperplasia of smooth muscle cells and adventitial fibroblasts. Adrenergic trophic activity is augmented in systemic arteries by injury and altered shear stress, which are key pathogenic stimuli in hypoxic PH, and contributes to neointimal formation and flow-mediated hypertrophic remodeling. Here we examined whether norepinephrine stimulates growth of the pulmonary artery (PA) and whether this is augmented in PH. PA from normoxic and hypoxic rats [9 days of 0.1 fraction of inspired O(2) (Fi(O(2)))] was studied in organ culture, where wall tension, Po(2), and Pco(2) were maintained at values present in normal and hypoxic PH rats. Norepinephrine treatment for 72 h increased DNA and protein content modestly in normoxic PA (+10%, P < 0.05). In hypoxic PA, these effects were augmented threefold (P < 0.05), and protein synthesis was increased 34-fold (P < 0.05). Inferior thoracic vena cava from normoxic or hypoxic rats was unaffected. Norepinephrine-induced growth in hypoxic PA was dose dependent, had efficacy greater than or equal to endothelin-1, required the presence of wall tension, and was inhibited by alpha(1A)-AR antagonist. In hypoxic pulmonary vasculature, alpha(1A)-AR was downregulated the least among alpha(1)-AR subtypes. These data demonstrate that norepinephrine has trophic activity in the PA that is augmented by PH. If evident in vivo in the pulmonary vasculature, adrenergic-induced growth may contribute to the vascular hyperplasia that participates in hypoxic PH. 相似文献
995.
Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke 总被引:1,自引:0,他引:1
Hong H Zeng JS Kreulen DL Kaufman DI Chen AF 《American journal of physiology. Heart and circulatory physiology》2006,291(5):H2210-H2215
Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. 相似文献
996.
997.
998.
Characterization of a fungal strain capable of degrading chlorpyrifos and
its use in detoxification of the insecticide on vegetables 总被引:2,自引:0,他引:2
A fungal strain capable of utilizing chlorpyrifos as sole carbon and energy sources was isolated from soil by enrichment cultivation approach. The half-lives of degradation (DT50) for chlorpyrifos at concentrations of 1, 10, and 100 mg l−1 by the fungal strain DSP in mineral salt medium were measured to be 2.03, 2.93, and 3.49 days, respectively. Two cell-free extracts [E (1:10) and E (1:20)] from the fungal strain DSP in bran–glucose medium were prepared and used to enhance chlorpyrifos degradation on vegetables. Compared with the controls, the DT50 of chlorpyrifos were reduced by 70.3%, 65.6%, 80.6%, 80.6%, and 86.1%, and by 53.8%, 43.2%, 66.0%, 54.3%, and 67.7% on E (1:20) and E (1:10) treated pakchoi, water spinach, Malabar spinach, haricot beans, and pepper, respectively. The 7-day residual values (R
7) of chlorpyrifos on E (1:10) treated vegetables were all lower than the corresponding maximum residue levels of European Union (EU MRLs), except that the R
7 value on haricot beans was slightly higher than the corresponding EU MRLs. The results indicate that cell-free extracts could rapidly degrade chlorpyrifos residues on vegetables. 相似文献
999.
Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways
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Focal adhesion kinase family interacting protein of 200 kD (FIP200) has been shown to regulate diverse cellular functions such as cell size, proliferation, and migration in vitro. However, the function of FIP200 in vivo has not been investigated. We show that targeted deletion of FIP200 in the mouse led to embryonic death at mid/late gestation associated with heart failure and liver degeneration. We found that FIP200 knockout (KO) embryos show reduced S6 kinase activation and cell size as a result of increased tuberous sclerosis complex function. Furthermore, FIP200 KO embryos exhibited significant apoptosis in heart and liver. Consistent with this, FIP200 KO mouse embryo fibroblasts and liver cells showed increased apoptosis and reduced c-Jun N-terminal kinase phosphorylation in response to tumor necrosis factor (TNF) alpha stimulation, which might be mediated by FIP200 interaction with apoptosis signal-regulating kinase 1 (ASK1) and TNF receptor-associated factor 2 (TRAF2), regulation of TRAF2-ASK1 interaction, and ASK1 phosphorylation. Together, our results reveal that FIP200 functions as a regulatory node to couple two important signaling pathways to regulate cell growth and survival during mouse embryogenesis. 相似文献
1000.
The mechanism regulating primordial follicle formation remains largely unexplored because of the developmental particularity of female germ cells and their ultimate functional structure as follicles. Using an in vitro follicle reconstitution culture model, we explored, in the present study, the possibility of producing transgenetic follicles in vitro. We found that mouse fetal ovarian germ cells progressively lose the flexibility for gene manipulation with their oogonia-oocyte transformation upon entering meiosis, the borderline of which was at around embryonic age of 13.5 days post coitus (dpc). Interestingly, we further observed that fetal ovarian cells, only at this age or beyond achieve the capacity to reform the follicles in culture. Screening of well-known marker gene (Zp1-3, Figalpha, and Cx43) expression in cultured fetal ovarian cells of various developmental ages revealed that Figalpha is one of the determining factors for normal primordial follicle formation. By conducting reciprocal follicle reconstitution experiments, we provided further evidence that a synchronized germ-somatic cell interaction determines the normal duration of primordial folliculogenesis. Besides uncovering a potentially important regulatory mechanism for normal oocyte differentiation and follicle formation, this observation offers an alternative approach to produce transgenic oocytes/follicles, and thus animal models. 相似文献