Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.     

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.     

Sexual process in eight-spored ascus-forming yeast, Schizosaccharomyces japonicus II. Dependency of the sexual process on the carbon source

The carbon-source dependency of the sexual process in Schizosaccharomycesjaponicus was studied. Schiz. japonicus grew well in vegetativemedia containing glucose, sucrose, fructose or raffinose, anddid poorly in one containing mannose. On the other hand, itssexual process proceeded well in sporulation media containingglucose, sucrose or mannose, and was markedly delayed in thosecontaining fructose or raffinose. Neither vegetative growthnor sexual process occurred when non-fermentable carbon sources,such as glycerol, were used. The amount of glucose in the sporulationmedium sufficient for completion of the sexual process varieddepending on the cell-population density. Glucose was requiredfor both zygote and ascus formation but not for spore liberation.Cells were committed to sporulation shortly after the stageof zygote formation. (Received August 3, 1978; )     

LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins

Neurons are polarized cells that contain distinct sets of proteins in their axons and dendrites. Synaptic vesicles (SV) and many SV proteins are exclusively localized in the presynaptic regions but not in dendrites. Despite their fundamental importance, the mechanisms underlying the polarized localization of SV proteins remain unclear. The transparent nematode Caenorhabditis elegans can be used to examine sorting and transport of SV proteins in vivo. Here, we identify a novel protein kinase LRK-1, a C. elegans homolog of the familial Parkinsonism gene PARK8/LRRK2 that is required for polarized localization of SV proteins. In lrk-1 deletion mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons. This aberrant localization of SV proteins in the dendrites is dependent on the AP-1 mu1 clathrin adaptor UNC-101, which is involved in polarized dendritic transport, but not on UNC-104 kinesin, which is required for the transport of SV to presynaptic regions. The LRK-1 proteins are localized in the Golgi apparatus. These results suggest that the LRK-1 protein kinase determines polarized sorting of SV proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi.     

Safety of intradermal injection of monosodium urate crystals as a vaccine carrier in volunteers

Monosodium urate (MSU) crystals are known to induce gouty arthritis, but also evoke specific cell immunity and work as an adjuvant by delivering several kinds of binding proteins, including idiotypic cancer vaccine peptides into dendritic cells. To investigate the potency of MSU crystals as a cancer vaccine carrier in vivo, this preclinical study examined whether intradermal injection of MSU crystals was safe for healthy adults. Subjects comprised 12 volunteers. Four different dose levels of MSU crystals were injected as follows: 2 μg (n = 3), 20 μg (n = 3), 200 μg (n = 3), or 2000?μg (n = 3). At 24 hours after administration, documented erythema was seen around the injection site in a dose-dependent manner, particularly in all adults with MSU dose ≥200 μg. However, redness was limited to the grade I level of the National Cancer Institute toxicity criteria. Serum uric acid levels did not show any change before and after injection. Moreover, neither gouty arthritis nor tophi developed in any volunteers, indicating that intradermal injection of MSU crystals did not induce systemic inflammation at the doses that evoked significant local inflammation. These findings suggest that intradermal injection of MSU crystals is fundamentally safe and should be made available for clinical trials using MSU-crystal-conjugated cancer vaccines.     

An FXPRLamide neuropeptide induces seasonal reproductive polyphenism underlying a life-history tradeoff in the tussock moth

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH(2) (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.     

A novel homocysteine-responsive gene, smap8, modulates mitogenesis in rat vascular smooth muscle cells.

We isolated the cDNA of a gene, designated smooth muscle-associated protein 8 (smap8), during a search for new genes expressed in human aortic smooth muscle cells. The full-length smap8 cDNA is 3241 bp long and contains an open reading frame of 1113 bp encoding an approximately 45 kDa soluble protein identical to NDRG4 protein. Smap8 mRNA was expressed predominantly in the brain and heart, and moderately in vascular smooth muscle cells. Expression of smap8 mRNA was induced within 3-12 h by treatment with 10 mm homocysteine in rat aortic smooth muscle cells (A10 cells). Expression of exogenous smap8 markedly reduced both the proliferation and migration rates of rat A10 cells, however, PDGF-induced proliferation was significantly enhanced in smap8-expressed cells compared with mock-transfected cells. To ascertain the involvement of smap8 in mitogenesis, we tested the effects of stimulation of smap8, MEK1/2 or ERK1/2, which is known as a proliferation relating intermediate, by various growth factors and cytokines. PDGF was the most prominent in promoting phosphorylation of the smap8 protein. PDGF-dependent phosphorylation of smap8 was induced prior to ERK1/2 activation, and was repressed by staurosporine, a general inhibitor of serine/threonine kinases. Furthermore, activation of both MEK1/2 and ERK1/2 was markedly enhanced in these cells. Smap8 might therefore regulate the potentiation of ERK1/2 signalling induced by PDGF treatment. Our results imply that smap8 is involved in the regulation of mitogenic signalling in vascular smooth muscle cells, possibly in response to a homocysteine-induced injury.     

Phi value analysis of an allosteric transition of GroEL based on a single-pathway model

There are currently two contradictory models for the kinetics of the ATP-induced GroEL allosteric transition occurring around 20 microM ATP. One model, proposed by Horovitz et al. demonstrates the existence of two parallel pathways for the allosteric transition and an abrupt ATP-dependent switch from one pathway to the other. The other model, which was proposed by the present authors, shows no need to assume the parallel pathways, and a combination of the transition-state theory and the Monod-Wyman-Changeux model of allostery can explain the kinetics as well as the equilibrium of the transition. The discrepancy appears to be due to whether we regard the transition as reversible or irreversible. Thus, here we have investigated the reversibility of the allosteric transition between 0 microM and 70 microM ATP by the use of a stopped-flow double-jump technique, which has allowed us to monitor the kinetics of the reverse reaction from the relaxed state at a high ATP concentration to the tense state at a low ATP concentration. The tryptophan fluorescence of a tryptophan-inserted variant of GroEL was used to follow the kinetics. As a result, the allosteric transition was shown to be a reversible process, supporting the validity of our model. We also show that the structural environment around the ATP-binding site of GroEL in the transition state is very similar to that in the relaxed state (Phi=0.9) by using a Phi value analysis in the kinetic Monod-Wyman-Changeux model, which is analogous to the mutational Phi value analysis in protein folding.     

Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.