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1.
Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2Andante to form aggregates with completely blocked active sites may contribute to this phenotype.  相似文献   
2.
We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca2+ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca2+]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca2+ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca2+ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.  相似文献   
3.
We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.  相似文献   
4.
Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.  相似文献   
5.
In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn2 +, Fe2 +, Mn2 +, Co2 + and Ni2 + homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn2 +. Incubation of ΔsmyiiP and WT cells with sub-lethal Mn2 + concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn2 + were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn2 + was increased by imposing a transmembrane H+ gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn2 + homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn2 + by a mechanism involving the proton motive force.  相似文献   
6.
β-Adrenergic agonists (β-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of β-agonists, a β2-adrenergic receptor (β2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant β2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of β-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized β2-AR proteins by β-agonists. The IC50 and limit of detection values for ractopamine were 30.38 μg L−1 and 5.20 μg L−1, respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of β-agonists in animal feeds.  相似文献   
7.
Collagenous and non-collagenous proteins (NCPs) in the extracellular matrix, as well as the coupling mechanisms between osteoclasts and osteoblasts, work together to ensure normal bone metabolism. Each protein plays one or more critical roles in bone metabolism, sometimes even contradictory, thus affecting the final mechanical, physical and chemical properties of bone tissue. Anomalies in the amount and structure of one or more of these proteins can cause abnormalities in bone formation and resorption, which consequently leads to malformations and defects, such as osteoporosis (OP). The connections between key proteins involved in matrix formation and resorption are far from being elucidated. In this review, we resume knowledge on the crosstalk between collagen type I and selected NCPs (Transforming Growth Factor-β, Insulin-like Growth Factor-1, Decorin, Osteonectin, Osteopontin, Bone Sialoprotein and Osteocalcin) of bone matrix, focusing on their possible involvement and role in OP. The different elements of this network can be pharmacologically targeted or used for the design/development of innovative regenerative strategies to modulate a feedback loop in bone remodelling.  相似文献   
8.
Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.  相似文献   
9.
Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.  相似文献   
10.
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