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41.
A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero‐length oxidative crosslinking 下载免费PDF全文
Jackie A. Thompson Owen W. Nadeau Gerald M. Carlson 《Protein science : a publication of the Protein Society》2015,24(12):1956-1963
Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)4 enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory β‐subunits to self‐associate. Here we report the effect of a peptide mimetic of the phosphoryltable N‐termini of β on the selective, zero‐length, oxidative crosslinking of these regulatory subunits to form β–β dimers in the nonactivated PhK complex. This peptide stimulated β–β dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of β competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic γ‐subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N‐termini of β and the regulatory C‐terminal domains of the γ‐subunits; phosphorylation of β weakens this interaction, leading to activation of the γ‐subunits. 相似文献
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Demery ZP Chappell J Martin GR 《Proceedings. Biological sciences / The Royal Society》2011,278(1725):3687-3693
Parrots are exceptional among birds for their high levels of exploratory behaviour and manipulatory abilities. It has been argued that foraging method is the prime determinant of a bird's visual field configuration. However, here we argue that the topography of visual fields in parrots is related to their playful dexterity, unique anatomy and particularly the tactile information that is gained through their bill tip organ during object manipulation. We measured the visual fields of Senegal parrots Poicephalus senegalus using the ophthalmoscopic reflex technique and also report some preliminary observations on the bill tip organ in this species. We found that the visual fields of Senegal parrots are unlike those described hitherto in any other bird species, with both a relatively broad frontal binocular field and a near comprehensive field of view around the head. The behavioural implications are discussed and we consider how extractive foraging and object exploration, mediated in part by tactile cues from the bill, has led to the absence of visual coverage of the region below the bill in favour of more comprehensive visual coverage above the head. 相似文献
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Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells 总被引:1,自引:0,他引:1
Doyon JB Zeitler B Cheng J Cheng AT Cherone JM Santiago Y Lee AH Vo TD Doyon Y Miller JC Paschon DE Zhang L Rebar EJ Gregory PD Urnov FD Drubin DG 《Nature cell biology》2011,13(3):331-337
Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics. 相似文献
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Chakravorty A Awad MM Hiscox TJ Cheung JK Carter GP Choo JM Lyras D Rood JI 《PloS one》2011,6(7):e22762
Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process. 相似文献
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We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells. 相似文献
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Tamminga C Sedegah M Regis D Chuang I Epstein JE Spring M Mendoza-Silveiras J McGrath S Maiolatesi S Reyes S Steinbeiss V Fedders C Smith K House B Ganeshan H Lejano J Abot E Banania GJ Sayo R Farooq F Belmonte M Murphy J Komisar J Williams J Shi M Brambilla D Manohar N Richie NO Wood C Limbach K Patterson NB Bruder JT Doolan DL King CR Diggs C Soisson L Carucci D Levine G Dutta S Hollingdale MR Ockenhouse CF Richie TL 《PloS one》2011,6(10):e25868
Background
A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.Methodology/Principal Findings
NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.Significance
The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.Trial Registration
ClinicalTrials.gov NCT00392015相似文献49.
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