Self-assembly and Self-replication of Short Amphiphilic β-sheet Peptides

Most self-replicating peptide systems are made of α-helix forming sequences. However, it has been postulated that shorter and simpler peptides may also serve as templates for replication when arranged into well-defined structures. We describe here the design and characterization of new peptides that form soluble β-sheet aggregates that serve to significantly accelerate their ligation and self-replication. We then discuss the relevance of these phenomena to early molecular evolution, in light of additional functionality associated with β-sheet assemblies.     

FGF signalling inhibits neural induction in human embryonic stem cells

Human embryonic stem cells (hESCs) can exit the self-renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self-renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non-neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.     

Sts-2 is a phosphatase that negatively regulates zeta-associated protein (ZAP)-70 and T cell receptor signaling pathways

T cell activity is controlled in large part by the T cell receptor (TCR). The TCR detects the presence of foreign pathogens and activates the T cell-mediated immune reaction. Numerous intracellular signaling pathways downstream of the TCR are involved in the process of T cell activation. Negative regulation of these pathways helps prevent excessive and deleterious T cell responses. Two homologous proteins, Sts-1 and Sts-2, have been shown to function as critical negative regulators of TCR signaling. The phosphoglycerate mutase-like domain of Sts-1 (Sts-1(PGM)) has a potent phosphatase activity that contributes to the suppression of TCR signaling. The function of Sts-2(PGM) as a phosphatase has been less clear, principally because its intrinsic enzyme activity has been difficult to detect. Here, we demonstrate that Sts-2 regulates the level of tyrosine phosphorylation on targets within T cells, among them the critical T cell tyrosine kinase Zap-70. Utilizing new phosphorylated substrates, we demonstrate that Sts-2(PGM) has clear, albeit weak, phosphatase activity. We further pinpoint Sts-2 residues Glu-481, Ser-552, and Ser-582 as specificity determinants, in that an Sts-2(PGM) triple mutant in which these three amino acids are altered to their counterparts in Sts-1(PGM) has substantially increased activity. Our results suggest that the phosphatase activities of both suppressor of TCR signaling homologues cooperate in a similar but independent fashion to help set the threshold for TCR-induced T cell activation.     

Heme interacts with c1q and inhibits the classical complement pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.     

Possible roles of exceptionally conserved residues around the selectivity filters of sodium and calcium channels

In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184-197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra- and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose π-electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outer-pore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.     

Synthesis and biological activity of 5-methylidene 1,2-dihydrochromeno[3,4-f]quinoline derivatives as progesterone receptor modulators

A series of 5-methylidene 1,2-dihydrochromeno[3,4-f]quinoline derivatives were synthesized and tested in biological assays to evaluate scope and limitations of the nonsteroidal SPRM pharmacophore (3). A number of orally available highly potent nonsteroidal modulators were identified by modification of the substituents at 5-methylidene position.     

Inhibition of human chymase by 2-amino-3,1-benzoxazin-4-ones

A series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chymase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase. Introduction of an aromatic moiety into the 2-substituent resulted in strong inhibition of chymase, cathepsin G, and chymotrypsin. Extension of the N(Me)CH2Ph substituent by one methylene unit was unfavourable to inhibit these proteases. Towards chymase, 2-(N-benzyl-N-methylamino)-4H-3,1-benzoxazin-4-one (32) and 2-(N-benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-4-one (33) were found to exhibit Ki values of 11 and 17 nM, respectively, and form stable acyl-enzymes with half-lives of 53 and 25 min, respectively. Benzoxazinone 33 also inhibited the human chymase-catalyzed formation of angiotensin 11 from angiotensin I.     

Characterization of leaf senescence and pod development in soybean explants

Excised soybean (Glycine max [L.] Merrill) cv Anoka leaf discs tend to remain green even after the corresponding intact leaves have turned yello on fruiting plants. We have found that explants which include a leaf along with a stem segment (below the node) and one or more pods (maintained on distilled H₂O) show similar but accelerated leaf yellowing and abscission compared with intact plants. In podded explants excised at pre-podfill, the leaves begin to yellow after 16 days, whereas those excised at late podfill begin to yellow after only 6 days. Although stomatal resistances remain low during the first light period after excision, they subsequently increase to levels above those in leaves of intact plants. Explants taken at mid to late podfill with one or more pods per node behave like intact plants in that pod load does not affect the time lag to leaf yellowing. Explant leaf yellowing and abscission are delayed by removal of the pods or seeds or by incubation in complete mineral nutrient solution or in 4.6 micromolar zeatin. Like chorophyll breakdown, protein loss is accelerated in the explants, but minerals or especially zeatin can retard the loss. Pods on explants show rates and patterns of color change (green to yellow to brown) similar to those of pods on intact plants. These changes start earlier in explants on water than in intact plants, but they can be delayed by adding zeatin. Seed dry weight increased in explants, almost as much as in intact plants. Explants appear to be good analogs of the corresponding parts of the intact plant, and they should prove useful for analyzing pod development and mechanisms of foliar senescence. Moreover, our data suggest that the flux of minerals and cytokinin from the roots could influence foliar senescence in soybeans, but increased stomatal resistance does not seem to cause foliar senescence.