Precise location of proton of beta-secretase for catalytic aspartates(Asp 32 and Asp 228)in Alzheimer’s patients

BACKGROUND：β-secretase （β-site APP cleavage rate-limiting enzyme, BACE） has been proposed as a promising therapeutic target for Alzheimer＇s disease （AD）. BACE inhibition reduces production of β-amyloid peptide （Aβ） and promotes neural regeneration. Two catalytic aspartates （Asp 32 and Asp 228） exist in a monoprotonated state in the active BACE site, but the precise proton location remains unclear.OBJECTIVE：To explore the entire process of BACE enzymatic hydrolysis using quantum chemistry calculations, and to identify the precise proton location for Asp 32 and Asp 228 during the enzymatic process.DESIGN, TIME AND SETTING：According to protonation state of BACE, four tautomers were designed and quantum chemistry calculations were performed at the Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, China between October 2008 and March 2009.MATERIALS：Hardware：linux workstation （Department of Equipment, Sun Yat-sen University, China）; software：QSITE, Glide, Maestro （Schrodinger LLC, USA）, MOPAC 2007 （CAChe Research LLC, USA）, Triton 4.0 （National Centre for Biomolecular Research, Czech Republic） were used.METHODS：Using crystal structures of BACE to build a catalytic model （enzyme, catalytic water, and substrate peptide EVNLAAEF） on the computer and superimposition, four BACE tautomers （32i, 320, 228i, and 2280） in the monoprotonated state were developed with Schrodinger package. Hybrid quantum mechanical/molecular mechanic （QM/MM） calculations were performed at the B3LYP density functional theory level to identify the precise proton location for the dyad aspartic residues （Asp 32 and Asp 228）. Using the most possible tautomer as the reactant, the entire enzymatic hydrolysis of substrate EVNL/AAEF was simulated at the semiempirical level.MAIN OUTCOME MEASURES：The precise proton location of was measured by analyzing co-planarities of 4 BACE tautorners （32i, 32o, 228i, and 2280） in the monoprotonated state, because the dihedral formed by the carboxyl oxygen atoms of the dyad aspartic residues. The transition state and the production state, as well as activation energies and reaction enthalpies, were measured by calculating geometric and energy changes during catalytic reaction of the system.RESULTS：In the 2280 BACE tautomer, the dihedral angle of the four oxygen atoms in the catalytic aspartates was 8.7°, which was the lowest of four tautomers. The lowest activation energy and highest reaction enthalpy （Ea = 216.30 kJ/mol, AH = 30.98 kJ/mol） were also found in 2280, among the four tautomers during the catalytic reaction. In addition, when the reaction proceeded to the transition state, followed by product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i） from the outer oxygen of Asp 228 （228o）.CONCLUSION：Results demonstrated the mechanism of Aβ generation. At beginning of BACE catalytic reaction, the precise proton location was preferred on the outer oxygen of Asp 228 （228o）. In this protonation state, catalytic reaction can proceed smoothly, with reduced active energy and heat release. When the reaction proceeded to the transition state and product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i）. These results provide theoretical guidance for designing new drugs to protect neural cells and promote neural regeneration in Alzheimer＇s patients.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

X-ray diffraction enhanced imaging study of intraocular tumors in human beings

Diffraction enhanced imaging （DEI） with edge enhancement is suitable for the observation of weakly absorbing objects. The potential ability of the DEI was explored for displaying the microanatomy and pathology of human eyeball in this work. The images of surgical specimens from malignant intraocular tumor of hospitalized patients were taken using the hard X-rays from the topography station of Beamline 4W1A at Beijing Synchrotron Radiation Facility （BSRF）. The obtained radiographic images were analyzed in correlation with those of pathology. The results show that the anatomic and pathologic details of intraocular tumors in human beings can be observed clearly by DEI for the first time, with good visualization of the microscopic details of eyeball ring such as sclera, choroids and other details of intraocular organelles. And the best resolution of DEI images reaches up to the magnitude of several tens of μm. The results suggest that it is capable of exhibiting clearly the details of intraocular tumor using DEI method.