Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

Integrin αIIbβ3: A novel effector of Gα13

Under physiological conditions, circulating platelets are discoid in shape.¹ On these platelets, the fibrinogen receptor (integrin α_IIbβ₃) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin α_IIbβ₃ through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.² Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin α_IIbβ₃, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin α_IIbβ₃. Platelets express nine Gα subunits; namely G_q, G_i1, G_i2, G_i3, G_z, G₁₂, G₁₃, G_s and G₁₆. Previous studies have shown that a small G-protein, RhoA, is activated by the G_12/13 family and plays a crucial role in calcium-independent platelet shape change.³ However, RhoA is also activated by α_IIbβ₃ and inhibits platelet spreading to trigger clot retraction.⁴ Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα₁₃ and α_IIbβ₃.⁵By generating mice in which the platelets were depleted of Gα₁₃ using siRNA technology, Gong et al. investigated the role of Gα₁₃-mediated signaling on platelet spreading on immobilized Fg.⁵ The confocal images very clearly showed that, in the absence of Gα₁₃, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin α_IIbβ₃ inhibits platelet spreading. Interestingly, Gα₁₃-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα₁₃ is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.⁵Since Gα₁₃ regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα₁₃ with α_IIbβ₃ using co-immunoprecipitation and GST pull-down assays.⁵ They found that the GTP-bound form of Gα₁₃ shows enhanced interaction with the integrin β₃ subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα₁₃ and β₃ cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.⁵ These findings indicate that Gα₁₃ is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα₁₃ (Fig. 1A). This GTP-bound Gα₁₃ interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα₁₃ and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα₁₃ during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα₁₃ leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to α_IIbβ₃. Active Gα13 binds to the cytoplasmic domain of β₃ leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β₃ cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin α_IIbβ₃ as an effector of Gα₁₃. The study also convincingly shows that Gα₁₃ bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα₁₃ in an in vivo setting using siRNA represents a technological advancement. Since Gα₁₃ binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα₁₃, as there are far less Gα₁₃ molecules in a single platelet than the number of α_IIbβ₃ molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα₁₃ is associated with β₃ in resting platelets, which requires some explanation.⁵ It is also not clear if Gα₁₃ remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα₁₃ in order to achieve efficient platelet spreading and clot retraction.     

The RSC chromatin remodelling ATPase translocates DNA with high force and small step size

ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ～35 bp, a speed of ～25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force.     

Physiology,Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.     

Molecular docking studies of anti-apoptotic BCL-2, BCL-XL, and MCL-1 proteins with ginsenosides from Panax ginseng

Anti-apoptotic proteins such as BCL-2, BCL-XL and MCL-1 bind with pro-apoptotic proteins to induce apoptosis mechanism. BCL-2 family proteins are key regulators of apoptosis process. Over expression of these anti-apoptotic proteins lead to several cancers by preventing apoptosis. A number of studies revealed that ginseng derivatives reduce tumor growth. Ginseng, the most valuable medicinal herb found in eastern Asia belongs to Araliaceae family. In this study, docking simulations were performed for anti-apoptotic proteins with several ginsenosides from Panax ginseng. Our finding shows ginsenosides Rf, Rg1, Rg3 and Rh2 have more binding affinity with BCL-2, BCL-XL and MCL-1 and other ginsenosides also interact with each anti-apoptotic proteins. Therefore, ginseng derivatives represent a novel class of potent inhibitors and could be used for cancer chemotherapy.