Predictive Potential of Circulating Ube2h mRNA as an E2 Ubiquitin-Conjugating Enzyme for Diagnosis or Treatment of Alzheimer's Disease.

Neurodegenerative disorders are caused by neuronal cell death, miscommunications between synapse, and abnormal accumulations of proteins in the brain. Alzheimer's disease (AD) is one of the age-related disorders, which are the most common degenerative disorders today, and strongly affects memory consolidation and cognitive function in the brain. Amyloid-β and tau proteins are triggers for AD pathogenesis, and usually used as AD candidate biomarkers in the clinical research. Especially, clinical exam, brain imaging and molecular biological methods are being used to diagnosis for AD. Genome-wide association study (GWAS) is a new biomedical method, and its use contributes to understanding many human diseases, including brain diseases. Here, we identified ubiquitin conjugating enzyme E2 (Ube2) gene expression in neurons through GWAS. The subfamilies of Ube2's genetic expression and inborn errors affect the ubiquitin proteasome system (UPS), leading to protein degradation in the brain. We found that only Ube2h mRNA transcription was significantly increased in the blood from AD, however we did not find any change of Ube2 subfamily genes' expression in the blood and brain tissue. These data may provide information for diagnosis or clinical approach, and suggest that cell-free circulating Ube2h mRNA is a novel potential biomarker for AD.

software [3] using the Amber99sb force field and the TIP3P water model [4]. The NDM-1-drug system was first energy-relaxed with 2000 steps of the steepest-descent energy minimization followed by another 2000 steps of conjugate-gradient energy minimization. The system was then equilibrated by a 500 ps molecular dynamic run with positional restraints on both the protein and the ligand in order to allow relaxation of the solvent molecules. The first equilibration run was followed by a 160 ns MD run without position restraints on the solute.
The first 30 ns of the trajectory were not used in the subsequent analysis for minimization of convergence artefacts. Equilibration of the trajectory was checked by monitoring the equilibration of quantities such as the root-mean-square deviation (RMSD) with respect to the initial structure, the internal protein energy, and the fluctuations calculated for different time intervals. The electrostatic term was described with the particle mesh Ewald algorithm. The LINCS [5] algorithm was used to constrain all bond lengths. For the water molecules, the SETTLE algorithm [5] was used. The dielectric permittivity was set as 1 and a time step of 2 fs was used. All atoms were given an initial velocity determined from the Maxwell distribution at the desired initial temperature of 300 K. The density of the system was adjusted during the first equilibration runs at NPT conditions by weak coupling to a bath of constant pressure (P0 = 1 bar, coupling time P = 0.5 ps) [6]. In all simulations, the temperature was maintained close to the intended values by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The proteins and the rest of the system were coupled separately to the temperature bath. The structural cluster analysis was carried out by using the method described by Daura and co-workers with a cutoff of 0.25 nm [6].
The parameters of ZINC05683641 were estimated with the antechamber program [7] and the AM1-BCC partial atomic charges from the Amber suite [8]. Analysis of the trajectories was performed using the VMD, PyMOL, and Gromacs analysis tools.

Calculation of the binding free energy
In this work, the binding free energies have been calculated using the MM-GBSA approach [9,10] supplied with the Amber 10 package. We chose a total number of 100 snapshots evenly from the last 10 ns on the MD trajectory with an interval of 10 ps. The MM-GBSA method can be conceptually summarized as follows: (1) where EMM is the summation of the van der Waals ( Evdw) and the electrostatic ( Eele) interaction energies.
In addition, Gsol, which denotes the solvation free energy, can be computed as the summation and two mutants NDM-1 with inhibitor were measured using the fluorescence-quenching method [12][13][14] .

Expression and purification of WT-PLY and mutants
Plasmids harboring the coding sequences for WT-NDM-1, W93A-NDM-1, and H250A-NDM-1 were transformed into E. coli BL21 (DE3). The E. coli strain was cultured in 1,000 mL of LB broth supplemented with 50 μg mL -1 kanamycin until the OD 600 nm of the cultures reached 0.6-0.8.
Protein expression was induced by using 0.3 mM IPTG for 12 h at 16 °C. The cells were harvested and washed once with sterile PBS. The pellets were suspended in PBS and lysed by sonication. The cell lysate was centrifuged at 12,000g for 30 min, and the supernatants were collected for the subsequent protein purification as described by Liu et.al [16].

Nitrocefin assay
A nitrocefin assay was used for the identification of potentially effective inhibitors and for further determination of the inhibitory effect of ZINC05683641 on the hydrolysis activities of NDM-1. The initial identifying of NDM-1 inhibitory activities was carried out on the compounds at a concentration of 1 mM (Table 1), and the compounds with greater than 50% inhibition at 1 mM were further tested to obtain their IC50 values against NDM-1. Nitrocefin serves as an indicator whose colour changes from yellow to red with increased hydrolysis. antagonism [17].

Materials
Meropenem, nitrocefin and 20 compounds from virtual screening were purchased from Sigma-Aldrich (St. Louis, MO, USA).