The amino-terminal helix modulates light-activated conformational changes in AsLOV2

The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.     

The number, age, sharing and relatedness of S-locus specificities in Prunus.

In gametophytic self-incompatibility systems, many specificities (different 'lock-and-key' combinations) are maintained by frequency-dependent selection for very long evolutionary times. In Solanaceae, trans-specific evolution (the observation that an allele from one species may be more closely related to an allele from another species than to others from the same species) has been taken as an argument for the very old age of specificities. In this work, by determining, for the first time, the age of extant Prunus species, we show that this reasoning cannot be applied to Prunoideae. Furthermore, since our sample size is large (all S-RNase encoding the female component and SFB encoding the male component GenBank sequences), we were able to estimate the age of the oldest Prunus specificities. By doing so, we show that the lower variability levels at the Prunus S-locus, in comparison with Solanaceae, is due to the younger age of Prunus alleles, and not to a difference in silent mutation rates. We show that the ancestor to extant Prunus species harboured at least 102 specificities, in contrast to the maximum of 33 observed in extant Prunus species. Since the number of specificities that can be maintained in a population depends on the effective population size, this observation suggests a bottleneck in Prunus evolutionary history. Loss of specificities may have occurred during this event. Using only information on amino acid sites that determine specificity differences, and a simulation approach, we show that a model that assumes closely related specificities are not preferentially lost during evolution, fails to predict the observed degree of specificity relatedness.     

α-Guanidinoglutaric Acid, an Endogenous Convulsant, as a Novel Nitric Oxide Synthase Inhibitor

Abstract: The effects of α-guanidinoglutaric acid (GGA), the levels of which were increased in the cobalt-induced epileptic focus tissue in the cerebral cortex of cats, on brain nitric oxide synthase (NOS) activity were observed. GGA inhibited NOS activity in a linear mixed manner ( K _i = 2.69 µ M ) and was as effective as N ^G-monomethyl- l -arginine (MeArg; K _i = 3.51 µ M ), a well-known NOS inhibitor. Although MeArg was synthesized by substituting the guanidino nitrogen of l -arginine (Arg), GGA was a non-guanidino nitrogen-substituted guanidino compound. On the other hand, Arg, which is an endogenous NOS substrate, elevates the threshold of seizures induced by GGA. There is evidence that GGA is an endogenous, potent, and non-guanidino nitrogen-substituted NOS inhibitor and that suppression of nitric oxide biosynthesis may be involved in GGA-induced convulsions. Therefore, GGA may be a useful tool in elucidating the chemical nature of NOS and the physiological function of nitric oxide.     

Involvement of fission yeast Clr6-HDAC in regulation of the checkpoint kinase Cds1

Modification of the N-terminal tail of histones is required for various nuclear processes. Here, we show that fission yeast Clr6-HDAC (histone deacetylase) regulates the checkpoint kinase Cds1 when DNA replication encounters a stressful condition. We found that the global level of acetylation of histone H4 was constant throughout the normal cell cycle, but was reduced significantly when the cell recovered from the HU-induced cell cycle arrest (or slow DNA replication). We identified the Clr6-HDAC as a component responsible for the reduction in the level of the H4 acetylation. Although DNA replication was completed, the HU-induced cell cycle arrest could not be released even after removal of HU in the clr6-1 mutant. Under this experimental condition, Cds1 kinase was maintained active and remained bound tightly to chromatin. We also demonstrated that Cds1 was active even after treatment with caffeine, an inhibitor for ATM/ATR that is an activator of Cds1. These results indicate that inactivation of Cds1 requires functional Clr6-HDAC independently of the conventional DNA replication checkpoint. When DNA replication is impeded, Clr6-HDAC activity may monitor damage on chromatin structure/environment, which is required for inactivation of Cds1.     

Identification of a novel retrovirus long terminal repeat (LTR) that is targeted by p51A (TAp63gamma) and selective dominant-negative activity of p73L (DeltaNp63alpha) toward p53-responsive promoter activities

The p51/p73L/p63/p40 gene, recently identified as a p53 homolog, encodes two major isoforms, p51A and p73L, which are suggested to have similar functions synonymous with p53 and dominant-negative activity toward both p53 and p51A, respectively. We have cloned a high affinity genomic fragment bound to p51A that was assigned to be a novel retrovirus long terminal repeat. Strikingly, this fragment was found to bind to both p53 and p73L with similar affinity to p51A. Additional demonstration with known p53 response elements suggested that DNA-binding profiles of p51A and p73L were very similar but were distinct from that of p53. Consistent with this novel finding, transient cotransfection experiments in mammalian cells suggested that p73L, when it was expressed at a low level, selectively suppressed p53-dependent transactivation of p21-luc and mdm2-luc but not of cyclinG-luc and bax-luc reporters. These data raise the possibility that p73L differentially modulates the p53 function according to the distinct DNA-binding affinity between these two proteins.     

Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI-1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI-1B at more than 1 ng/10 g total protein was significantly retarded compared to that on non-transgenic control plants.     

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.     

Successful shape-based virtual screening: the discovery of a potent inhibitor of the type I TGFbeta receptor kinase (TbetaRI)

We describe the discovery, using shape-based virtual screening, of a potent, ATP site-directed inhibitor of the TβRI kinase, an important and novel drug target for fibrosis and cancer. The first detailed report of a TβRI kinase small molecule co-complex confirms the predicted binding interactions of our small molecule inhibitor, which stabilizes the inactive kinase conformation. Our results validate shape-based screening as a powerful tool to discover useful leads against a new drug target.