Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0? in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.     

Among γ-secretase substrates Notch1 alone is sufficient to block neurogenesis but does not confer self-renewal properties to neural stem cells

Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.     

Working memory deficits in neuronal nitric oxide synthase knockout mice: potential impairments in prefrontal cortex mediated cognitive function

Neuronal nitric oxide synthase (nNOS) forms nitric oxide (NO), which functions as a signaling molecule via S-nitrosylation of various proteins and regulation of soluble guanylate cyclase (cGC)/cyclic guanosine monophosphate (cGMP) pathway in the central nervous system. nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function. Human genetics have identified nNOS and several downstream effectors of nNOS as risk genes for schizophrenia. Besides the disease itself, nNOS has also been associated with prefrontal cortical functioning, including cognition, of which disturbances are a core feature of schizophrenia. Although mice with genetic deletion of nNOS display various behavioral deficits, no studies have investigated prefrontal cortex-associated behaviors. Here, we report that nNOS knockout (KO) mice exhibit hyperactivity and impairments in contextual fear conditioning, results consistent with previous reports. nNOS KO mice also display mild impairments in object recognition memory. Most importantly, we report for the first time working memory deficits, potential impairments in prefrontal cortex mediated cognitive function in nNOS KO mice. Furthermore, we demonstrate Disrupted-in-Schizophrenia 1 (DISC1), another genetic risk factor for schizophrenia that plays roles for cortical development and prefrontal cortex functioning, including working memory, is a novel protein binding partner of nNOS in the developing cerebral cortex. Of note, genetic deletion of nNOS appears to increase the binding of DISC1 to NDEL1, regulating neurite outgrowth as previously reported. These results suggest that nNOS KO mice are useful tools in studying the role of nNOS signaling in cortical development and prefrontal cortical functioning.     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

Targeted disruption of Mcm10 causes defective embryonic cell proliferation and early embryo lethality

Minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein, which plays a key role in the initiation of eukaryotic chromosomal DNA replication and elongation. To elucidate the physiological importance of MCM10 in vivo, we generated conventional knockout mice. No MCM10-null embryos were recovered after E8.5, and the mutation was found to be lethal before the implantation stage. Mutant embryos showed apparently normal growth until the morula stage, but growth defects after this stage. The dramatic reduction of 5-bromo-2-deoxyuridine (BrdU) incorporation in the mutant embryo, followed by cell death, suggests that defective cell proliferation may underlie this developmental failure. Taken together, these findings provide the first unequivocal genetic evidence for an essential and non-redundant physiological role of MCM10 during murine peri-implantation development.     

Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation

Alzheimer’s disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid β (Aβ) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aβ aggregation, in N2aSwe. Intracerebroventricular injection of Aβ_25–35 in C57BL/6J mice resulted in increased immunoreactivity of Aβ and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aβ administration and once a day for 4 weeks post-surgery almost completely prevented the Aβ-induced increases of Aβ and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aβ administration, when Aβ was already accumulated, did not prevent the Aβ-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aβ peptide, but decreased ApoE levels in Aβ-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aβ-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aβ accumulation and improved memory impairment induced by Aβ_25–35. The beneficial effects of cilostazol might be explained by the reduction of Aβ accumulation and tau phosphorylation, not through an increase in Aβ degradation but via a significant decrease in ApoE-mediated Aβ aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.     

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is known to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X_L and Bcl-2. A structural model of the Bcl-X_L/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X_L/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X_L. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.     

The small GTPase Rac1 is involved in the maintenance of stemness and malignancies in glioma stem-like cells

A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem-like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem-like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1-Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self-renewal-related proteins and neurosphere formation. Moreover, down-regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.