Sagar H. Barage

Amyloid cascade hypothesis: Pathogenesis and therapeutic strategies in Alzheimer's disease

Alzheimers disease is an irreversible, progressive neurodegenerative disorder. Various therapeutic approaches are being used to improve the cholinergic neurotransmission, but their role in AD pathogenesis is still unknown. Although, an increase in tau protein concentration in CSF has been described in AD, but several issues remains unclear. Extensive and accurate analysis of CSF could be helpful to define presence of tau proteins in physiological conditions, or released during the progression of neurodegenerative disease. The amyloid cascade hypothesis postulates that the neurodegeneration in AD caused by abnormal accumulation of amyloid beta (Aβ) plaques in various areas of the brain. The amyloid hypothesis has continued to gain support over the last two decades, particularly from genetic studies. Therefore, current research progress in several areas of therapies shall provide an effective treatment to cure this devastating disease. This review critically evaluates general biochemical and physiological functions of Aβ directed therapeutics and their relevance.

Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide

Angiotensin converting enzyme (ACE) cleaves amyloid beta peptide. So far this cleavage mechanism has not been studied in detail at atomic level. Keeping this view in mind, we performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn2+ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide has been made to investigate the interactions of Aβ-peptide with enzyme tACE. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn2+. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient.

Structural analysis of membrane-bound hECE-1 dimer using molecular modeling techniques: insights into conformational changes and Aβ1–42 peptide binding

The human endothelin converting enzyme-1 (hECE-1) is a homodimer linked by a single disulfide bridge and has been identified as an important target for Alzheimer’s disease. Structural analysis of hECE-1 dimer could lead to design specific and effective therapies against Alzheimer’s disease. Hence, in the present study homology model of transmembrane helix has been constructed and patched with available crystal structure of hECE-1 monomer. Then, membrane-bound whole model of hECE-1 dimer has been developed by considering biophysical properties of membrane proteins. The explicit molecular dynamics simulation revealed that the hECE-1 dimer exhibits conformational restrains and controls total central cavity by regulating the degree of fluctuations in some residues (238–226) for substrate/product entrance/exit sites. In turn, conformational rearrangements of interdomain linkers as well as helices close to the inner surface are responsible for increasing total central cavity of hECE-1 dimer. Further, the model of hECE-1 dimer was docked with Aβ1–42 followed by MD simulation to investigate possible orientation and interactions of Aβ1–42 in catalytic groove of hECE-1 dimer. The free energy calculations exposed the stability of complex and helped us to identify key residues of hECE-1 involved in interactions with Aβ1–42 peptide. Hence, the present study might be useful to understand structural significance of membrane-bound dimeric hECE-1 to design therapies against Alzheimer’s disease.

Homology modeling, molecular docking and MD simulation studies to investigate role of cysteine protease from Xanthomonas campestris in degradation of Aβ peptide

Cysteine protease is known to degrade amyloid beta peptide which is a causative agent of Alzheimers disease. This cleavage mechanism has not been studied in detail at the atomic level. Hence, a three-dimensional structure of cysteine protease from Xanthomonas campestris was constructed by homology modeling using Geno3D, SWISS-MODEL, and MODELLER 9v7. All the predicted models were analyzed by PROCHECK and PROSA. Three-dimensional model of cysteine protease built by MODELLER 9v7 shows similarity with human cathepsin B crystal structure. This model was then used further for docking and simulation studies. The molecular docking study revealed that Cys17, His87, and Gln88 residues of cysteine protease form an active site pocket similar to human cathepsin B. Then the docked complex was refined by molecular dynamic simulation to confirm its stable behavior over the entire simulation period. The molecular docking and MD simulation studies showed that the sulfhydryl hydrogen atom of Cys17 of cysteine protease interacts with carboxylic oxygen of Lys16 of Aβ peptide indicating the cleavage site. Thus, the cysteine protease model from X. campestris having similarity with human cathepsin B crystal structure may be used as an alternate approach to cleave Aβ peptide a causative agent of Alzheimers disease.

Insight into molecular interactions of Aβ peptide and gelatinase from Enterococcus faecalis: a molecular modeling approach

Alzheimers disease is characterized by the presence of extracellular deposition of amyloid beta (Aβ) peptides. The Aβ levels are maintained by a suitable balance between its production and clearance via receptor-mediated cellular uptake and direct enzymatic degradation. However, decreased production of Aβ degrading enzymes and loss of Aβ degrading activity in the human brain initiates the process of accumulation of Aβ peptides. Mutations in amyloid beta peptides are also known to reduce clearance of Aβ peptides. So it becomes necessary to understand molecular interactions involved in the process of Aβ degradation in detail at the atomic level. Hence, in this study, we used molecular docking and molecular dynamics simulation techniques to explore molecular interactions between gelatinase from Enterococcus faecalis and Aβ peptide. The comparison between wild type and mutant Aβ peptides revealed that the docked complex of gelatinase–wild type Aβ peptide is more stable as compared to mutant complex. These results showed that the residue Glu 328 of gelatinase from E. faecalis might play a catalytic role by serving as a proton shuttle to cleave wild type Aβ peptide in between Leu 34–Met 35. Thus, the results obtained from this study might be useful to design new strategies against Aβ peptide degradation.

Exploring mode of phosphoramidon and Aβ peptide binding to hECE-1 by molecular dynamics and docking studies.

Human Endothelin converting enzyme (hECE-1) has been widely known for its involvement in hydrolyzing Aβ peptides at multiple sites. In the present study we have performed molecular dynamics (MD) simulation of crystal structure complex of hECE-1 and its inhibitor phosphoramidon with Zn ion to understand the dynamic behavior of active site residues. Root Mean Square Deviation (RMSD) results revealed that enzyme hECE-1 structure was highly stable throughout the simulation period. The L-leucyl-L-tryptophan moiety and N-phosphoryl moiety of phosphoramidon was found in the S1 and S2 pockets of hECE-1 respectively. The inhibitor was stabilized by hydrogen bonding interactions with residues Arg 145, Asn 566, Pro 731 and His 732 of hECE-1. Based on this information molecular docking of hECE- 1 crystal structure with three different structures of Aβ peptides has been performed. Zinc ion interacts with His 607(NE2), His 611(NE2), Glu 667 (OE1, OE2) and backbone oxygen atom of Phe 19 showing catalytic coordination between Aβ peptide and hECE-1. The unusual orientation of Aβ peptide residues affects hydrophobic interactions and hydrogen bonding network between hECE-1 and Aβ peptide. The molecular basis of amyloid beta peptide cleavage by hECE-1 could aid in designing enzyme based therapies to control Aβ peptide concentration in Alzheimers patient.

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis

Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

Characterization of the algC Gene Expression Pattern in the Multidrug Resistant Acinetobacter baumannii AIIMS 7 and Correlation with Biofilm Development on Abiotic Surface

Relative quantification of algC gene expression was evaluated in the multidrug resistant strain Acinetobacter baumannii AIIMS 7 biofilm (3 to 96 h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expression pattern with maximum 81.59-fold increase in biofilm cells versus 3.24-fold in planktonic cells (P < 0.05). Expression levels strongly correlated with specific biofilm stages (scale of 3 to 96 h), coinciding maximum at initial surface attachment stage (9 h) and biofilm maturation stage (48 h). Cloning, heterologous expression, and bioinformatics analyses indicated algC gene product as the bifunctional enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) of ∼53 kDa size, which augmented biofilms significantly in algC clones compared to controls (lacking algC gene), further localized by scanning electron microscopy. Moreover, molecular dynamics analysis on the three-dimensional structure of PMM/PGM (simulated up to 10 ns) revealed enzyme structure as stable and similar to that in P. aeruginosa (synthesis of alginate and lipopolysaccharide core) and involved in constitution of biofilm EPS (extracellular polymeric substances). Our observation on differential expression pattern of algC having strong correlation with important biofilm stages, scanning electron-microscopic evidence of biofilm augmentation taken together with predictive enzyme functions via molecular dynamic (MD) simulation, proposes a new basis of A. baumannii AIIMS 7 biofilm development on inanimate surfaces.

Homology modeling and molecular docking studies of ArnA protein from Erwinia amylovora: role in polymyxin antibiotic resistance

The resistivity of plant pathogen Erwinia amylovora against the polymyxin group of antibiotics is enhanced by modification of lipid A from lipopolysaccharide with 4-amino-4-deoxy L-arabinose (Ara4N) catalyzed by a bifunctional protein ArnA. ArnA is the first enzyme in the lipid A modification pathway with distinct dehydrogenase and transformylase domains which has been known in development of resistivity to polymyxin group of antibiotics. Thus, three dimensional structure of ArnA protein from Erwinia amylovora was constructed using homology modeling technique. The quality and reliability of the generated 3-D model was then assessed by different online available programs such as What if, PROCHECK, QMEAN, ProSA along with superimposition by UCSF Chimera. Sequence analysis study of ArnA protein from E. coli, Erwinia amylovora, Yersinia pestis, Ps. aeruginosa and Salmonella showed conserved domains with exact active site residues. Molecular docking study of ArnA protein with substrate UDP-GlcA and different inhibitors such as 5-formyl-5,6,7,8 tetrahydrofolate, leucovorin and 5-methyl tetrahydrofolate revealed similar binding pocket. The residues ASN492, ARG510, SER433 and ARG619 of ArnA protein are involved in interactions with inhibitors. Thus, this study could be useful to understand the proper binding mode of inhibitors to inhibit the lipid A modification pathway of ArnA protein from plant pathogen Erwinia amylovora.