The role of amyloids in Alzheimer's and Parkinson's diseases.

With varying clinical symptoms, most neurodegenerative diseases are associated with abnormal loss of neurons. They share the same common pathogenic mechanisms involving misfolding and aggregation, and these visible aggregates of proteins are deposited in the central nervous system. Amyloid formation is thought to arise from partial unfolding of misfolded proteins leading to the exposure of hydrophobic surfaces, which interact with other similar structures and give rise to form dimers, oligomers, protofibrils, and eventually mature fibril aggregates. Accumulating evidence indicates that amyloid oligomers, not amyloid fibrils, are the most toxic species that causes Alzheimer's disease (AD) and Parkinson's disease (PD). AD has recently been recognized as the 'twenty-first century plague', with an incident rate of 1% at 60 years of age, which then doubles every fifth year. Currently, 5.3 million people in the US are afflicted with this disease, and the number of cases is expected to rise to 13.5 million by 2050. PD, a disorder of the brain, is the second most common form of dementia, characterized by difficulty in walking and movement. Keeping the above views in mind, in this review we have focused on the roles of amyloid in neurodegenerative diseases including AD and PD, the involvement of amyloid in mitochondrial dysfunction leading to neurodegeneration, are also considered in the review.

Mechanisms of amyloid proteins aggregation and their inhibition by antibodies, small molecule inhibitors, nano-particles and nano-bodies.

Protein misfolding and aggregation can be induced by a wide variety of factors, such as dominant disease-associated mutations, changes in the environmental conditions (pH, temperature, ionic strength, protein concentration, exposure to transition metal ions, exposure to toxins, posttranslational modifications including glycation, phosphorylation, and sulfation). Misfolded intermediates interact with similar intermediates and progressively form dimers, oligomers, protofibrils, and fibrils. In amyloidoses, fibrillar aggregates are deposited in the tissues either as intracellular inclusion or extracellular plaques (amyloid). When such proteinaceous deposit occurs in the neuronal cells, it initiates degeneration of neurons and consequently resulting in the manifestation of various neurodegenerative diseases. Several different types of molecules have been designed and tested both in vitro and in vivo to evaluate their anti-amyloidogenic efficacies. For instance, the native structure of a protein associated with amyloidosis could be stabilized by ligands, antibodies could be used to remove plaques, oligomer-specific antibody A11 could be used to remove oligomers, or prefibrillar aggregates could be removed by affibodies. Keeping the above views in mind, in this review we have discussed protein misfolding and aggregation, mechanisms of protein aggregation, factors responsible for aggregations, and strategies for aggregation inhibition.

Comprehensive analysis of the molecular docking of small molecule inhibitors to the Aß1-40 peptide and its Osaka-mutant: insights into the molecular mechanisms of Aß-peptide inhibition.

Alzheimer's disease (AD) is the most common form of age-related neurodegeneration occurs because of deposition of proteins in the form of extracellular plaques containing aggregated amyloid beta (Aß) peptide and intracellular neurofibrillary tangles composed of aggregated microtubule-binding protein tau. Amyloid aggregation process can be enhanced by several familial AD-associated mutations in Aß peptide. In this study, we have unravelled the interactions of 40 small molecule inhibitors with the Osaka-mutant of Aß1-40 peptide at atomic level and characterized modes of their binding to mutant Aß by docking approaches. We have also compared docking energies of these inhibitors with Osaka-mutant with those previously determined for the wild-type and Iowa-mutant peptides and discussed in light of the peptide conformations and non-covalent interactions. We have also discussed inhibition mechanisms of these three peptides. Our analyses revealed that these small molecules can efficiently inhibit Osaka-mutant. The binding modes of drugs with these three peptides are markedly different and so are the mechanisms of inhibition of these three peptides. Overall analysis of the data reveals that binding energy of Iowa-mutant drug complex is lowest and most stable which is followed wild-type peptide-drug complex followed by Osaka-mutant drug complex.Communicated by Ramaswamy H. Sarma.

Nanoparticle formulations in the diagnosis and therapy of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common age-related diseases that occurs because of the deposition of amyloid fibrils in a form of extracellular plaques containing ß-amyloid peptide (Aß) and tangles are found as intracellular deposit in the brain made up of twisted strands of aggregated microtubule binding protein. Scores of small molecule inhibitors have been designed for the treatment of AD. However some of these drugs cannot pass through the brain-blood-barrier (BBB). To overcome this problem, various nanoparticles (NPs) or nanomedicines (NMs) have been synthesized. These nanoparticles exploit the existing physiological mechanisms of passing through the BBB, including receptor- and adsorptive-mediated transcytosis that facilitate the transcellular transport of nanoparticle from the blood to the brain. During the last decades, varieties of nanoparticles that differ in the composition have been developed, and they have the potential application in the diagnostics and therapy of AD. The most common NP formulations that have major impact in the diagnosis and therapy of AD include polymeric NPs (PPs), gold NPs, gadolinium NPs, selenium NPs, protein-based NPs, polysaccharide-based NPs, etc. The goal of this review is to provide discussion of the application of different types of NP formulations in the diagnosis and therapy of AD.

Molecular docking of Aß1-40 peptide and its Iowa D23N mutant using small molecule inhibitors: Possible mechanisms of Aß-peptide inhibition.

Alzheimer's disease (AD) is the most common form of neurodegenerative diseases, characterized by the deposition of Aß (amyloid beta) peptide. In this study, we have unravelled the interactions as well as anti amyloidogenic behaviour of 40 small molecule inhibitors with Aß1-40 peptide and Iowa mutant D23N-Aß115-42 peptide at atomic level and their modes of binding by docking approaches. The binding mode between wild type peptide and drug is distinctly different from the Iowa-mutant-peptide and drug. Here we proposed possible mechanisms of amyloid beta peptide inhibition by small molecule and prevent monomer-monomer interactions via at least three different mechanisms. In the first mechanism, four catechins efficiently interacted with the C-terminal region of peptides through hydrogen bonds and inhibited the peptides. This may lead to blockage of access of second molecule of Aß-peptide. Secondly, in the case Iowa mutant D23N-Aß15-42 peptide, same catechin form hydrogen bond with the important mutated Asn23 residue which acts as hydrogen bond donor and acceptor leading to tight binding of inhibitor with the peptide and may prevent monomer-monomer interactions. The third mechanism relies on the ability of drug molecules to mask hydrophobic residues of the peptide, thereby possibly inhibiting hydrophobic interactions between the two beta peptides.

Emerging Targets and Latest Proteomics Based Therapeutic Approaches in Neurodegenerative Diseases.

Protein homeostasis (proteostasis) is achieved by the interplay among various components and pathways inside a cell. Dysfunction in proteostasis leads to protein misfolding and aggregation which is ubiquitously associated with many neurodegenerative disorders, although the exact role of these aggregate in the pathogenesis remains unknown. Many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and others are characterized by the conversion of specific protein aggregates into protein inclusions and/or plaques in degenerating brains. Apart from the conventional disease specific proteins, such as amyloid-ß, α - synuclein, huntingtin protein, and prions that are known to aggregate, a number of other proteins play a vital role in aggravating the disease condition. In this review, we discuss the disease etiology, mechanism, the role of various pathways, molecular machinery including molecular chaperones, protein degradation pathways, and the active formation of inclusions in various neurodegenerative diseases. We also highlight the approaches, strategies, and methods that have been used for the treatment of these complex diseases over the years and the efforts that have potential in the near future.

Structure, Function of Serine and Metallo-ß-lactamases and their Inhibitors.

Antibiotic resistance in gram-negative bacteria has emerged as a major health threat that occurs because these bacteria actively produce ß-lactamases responsible for the inactivation of ß-lactam antibiotics. The first ß lactamase was reported in E. coli back in 1940, before the release of the first antibiotic penicillin in clinical settings. Later on, large numbers of ß-lactamases have been discovered in Gram-positive, Gram-negative bacteria as well as mycobacteria. Currently, numerous three-dimensional structures of serine and metallo-ß-lactamases have been solved. The serine ß-lactamases essentially consist of two structural domains (an all α and an α/ß domain) and the active site is located at the groove between the two domains. The catalysis of serine ß-lactamase proceeds via acylation and deacylation reactions. The three dimensional structure of metallo-ß-lactamases displayed a common four layer "αß/ßα" motif, with a central "ßß"- sandwich by Zn2+ ion(s), and two α-helices are located on the either side. The active site of metallo-ß-lactamases contain either 1 or 2 Zn2+ ions, which is coordinated to metal ligating amino acids and polarized water molecule(s) necessary for the hydrolysis of ß-lactam antibiotics. Keeping the above views in mind, in this review we have shed light on the current knowledge of the structures and mechanisms of catalysis of serine and metallo-ß-lactamases. Moreover, mutational studies on ß-lactamases highlight the importance of the active site residues and residues in the vicinity to the active site pocket in the catalysis. To combat bacterial infections more effeciently novel inhibitors of ß-lactamase in combination with antibiotics have been used which also form the theme of the review.

Structure of amyloid oligomers and their mechanisms of toxicities: Targeting amyloid oligomers using novel therapeutic approaches.

Protein misfolding is one of the leading causes of amyloidoses. Protein misfolding occurs from changes in environmental conditions and host of other factors, including errors in post-translational modifications, increase in the rate of degradation, error in trafficking, loss of binding partners and oxidative damage. Misfolding gives rise to the formation of partially unfolded or misfolded intermediates, which have exposed hydrophobic residues and interact with complementary intermediates to form oligomers and consequently protofibrils and fibrils. The amyloid fibrils accumulate as amyloid deposits in the brain and central nervous system in Alzheimer's disease (AD), Prion disease and Parkinson's disease (PD). Initial studies have shown that amyloid fibrils were the main culprit behind toxicity that cause neurodegenerative diseases. However, attention shifted to the cytotoxicity of amyloid fibril precursors, notably amyloid oligomers, which are the major cause of toxicity. The mechanism of toxicity triggered by amyloid oligomers remains elusive. In this review, we have focused on the current knowledge of the structures of different aggregated states, including amyloid fibril, protofibrils, annular aggregates and oligomers. Based on the studies on the mechanism of toxicities, we hypothesize two major possible mechanisms of toxicities instigated by oligomers of Aß (amyloid beta), PrP (prion protein) (106-126), and α-Syn (alpha-synuclein) including direct formation of ion channels and neuron membrane disruption by the increase in membrane conductance or leakage in the presence of small globulomers to large prefibrillar assemblies. Finally, we have discussed various novel innovative approaches that target amyloid oligomers in Alzheimer's diseases, Prion disease and Parkinson's disease.

The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach.

Protein glycation is initiated by a nucleophilic addition reaction between the free amino group from a protein, lipid or nucleic acid and the carbonyl group of a reducing sugar. This reaction forms a reversible Schiff base, which rearranges over a period of days to produce ketoamine or Amadori products. The Amadori products undergo dehydration and rearrangements and develop a cross-link between adjacent proteins, giving rise to protein aggregation or advanced glycation end products (AGEs). A number of studies have shown that glycation induces the formation of the ß-sheet structure in ß-amyloid protein, α-synuclein, transthyretin (TTR), copper-zinc superoxide dismutase 1 (Cu, Zn-SOD-1), and prion protein. Aggregation of the ß-sheet structure in each case creates fibrillar structures, respectively causing Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and prion disease. It has been suggested that oligomeric species of glycated α-synuclein and prion are more toxic than fibrils. This review focuses on the pathway of AGE formation, the synthesis of different types of AGE, and the molecular mechanisms by which glycation causes various types of neurodegenerative disease. It discusses several new therapeutic approaches that have been applied to treat these devastating disorders, including the use of various synthetic and naturally occurring inhibitors. Modulation of the AGE-RAGE axis is now considered promising in the prevention of neurodegenerative diseases. Additionally, the review covers several defense enzymes and proteins in the human body that are important anti-glycating systems acting to prevent the development of neurodegenerative diseases.

Studies on structure-based sequence alignment and phylogenies of beta-lactamases.

The ß-lactamases enzymes cleave the amide bond in ß-lactam ring, rendering ß-lactam antibiotics harmless to bacteria. In this communication we have studied structure-function relationship and phylogenies of class A, B and D beta-lactamases using structure-based sequence alignment and phylip programs respectively. The data of structure-based sequence alignment suggests that in different isolates of TEM-1, mutations did not occur at or near sequence motifs. Since deletions are reported to be lethal to structure and function of enzyme. Therefore, in these variants antibiotic hydrolysis profile and specificity will be affected. The alignment data of class A enzyme SHV-1, CTX-M-15, class D enzyme, OXA-10, and class B enzyme VIM-2 and SIM-1 show sequence motifs along with other part of polypeptide are essentially conserved. These results imply that conformations of betalactamases are close to native state and possess normal hydrolytic activities towards beta-lactam antibiotics. However, class B enzyme such as IMP-1 and NDM-1 are less conserved than other class A and D studied here because mutation and deletions occurred at critically important region such as active site. Therefore, the structure of these beta-lactamases will be altered and antibiotic hydrolysis profile will be affected. Phylogenetic studies suggest that class A and D beta-lactamases including TOHO-1 and OXA-10 respectively evolved by horizontal gene transfer (HGT) whereas other member of class A such as TEM-1 evolved by gene duplication mechanism. Taken together, these studies justify structure-function relationship of beta-lactamases and phylogenetic studies suggest these enzymes evolved by different mechanisms.

BFluenza: A Proteomic Database on Bird Flu.

UNLABELLED: Influenza A virus subtype H5N1, also known as "bird flu" has been documented to cause an outbreak of respiratory diseases in humans. The unprecedented spread of highly pathogenic avian influenza type A is a threat to veterinary and human health. The BFluenza is a relational database which is solely devoted to proteomic information of H5N1 subtype. Bfluenza has novel features including computed physico-chemical properties data of H5N1 viral proteins, modeled structures of viral proteins, data of protein coordinates, experimental details, molecular description and bibliographic reference. The database also contains nucleotide and their decoded protein sequences data. The database can be searched in various modes by setting search options. The structure of viral protein could be visualized by JMol viewer or by Discovery Studio. AVAILABILITY: The database is available for free at http://www.bfluenza.info.

Structural and functional analysis of NS1 and NS2 proteins of H1N1 subtype.

Influenza A virus (H1N1), a genetic reassortment of endemic strains of human, avian and swine flu, has crossed species barrier to human and apparently acquired the capability of human to human transmission. Some strains of H5N1 subtype are highly virulent because NS1 protein inhibits antiviral interferon α/ß production. Another protein NS2 mediates export of viral ribonucleoprotein from nucleus to the cytoplasm through export signal. In this paper, we have studied structure-function relationships of these proteins of H1N1 subtype and have determined the cause of their pathogenicity. Our results showed that non-conservative mutations slightly stabilized or destabilized structural domains of NS1 or NS1-dsRNA complex, hence slightly increased or decreased the function of NS1 protein and consequently enhanced or reduced the pathogenicity of the H1N1 virus. NS2 protein of different strains carried non-conservative mutations in different domains, resulting in slight loss of function. These mutations slightly decreased the pathogenicity of the virus. Thus, the results confirm the structure-function relationships of these viral proteins.

Structure function studies on different structural domains of nucleoprotein of H1N1 subtype.

Recent 2009 flu pandemic is a global outbreak of a new strain of influenza A virus subtype H1N1. The H1N1 virus has crossed species barrier to human and apparently acquired the capability to transmit this disease from human to human. The NP is a multifunctional protein that not only encapsidates viral RNA (vRNA), but also forms homo-oligomer and thereby maintains RNP structure. It is also thought to be the key adaptor for virus and host cell interaction. Thus, it is one of the factor that play a key role in the pathogenesis of influenza A virus infection. Therefore, to understand the cause of pathogenicity of H1N1 virus, we have studied the structure-function relationship of different domains of NP. Our results showed that conservative mutation in NP of various strains were pathogenic in nature. However, non-conservative mutation slightly abrogated oligomerization and was therefore less pathogenic. Our results also suggest that beside tail and body domain, head domain may also participate in an oligomerization process.

Genetic variants of alpha1-antitrypsin.

Alpha1-antitrypsin (alpha1-AT) is a 52 kDa sialoglycoprotein. The function of alpha1-antitrypsin is to protect the lower respiratory tract of lungs from proteolytic degradation by neutrophil elastase. Severe genetic deficiency of alpha1-AT is associated with early onset emphysema and liver diseases. alpha1-AT also exhibits anti-inflammatory activities independent of its protease inhibitor function. There are over 90 genetic variants of human alpha1-antitrypsin. These variants occur due to amino acid substitution / deletion which results in charge differences. Based on charge differences these variants have been identified by isoelectric focusing. The two most common deficiency variants are S and Z. The S variant migrates anodal to Z variant. The Z variant migrates most cathodal in isoelectric focusing, hence named Z. In Z variant, the beta-sheet A undergoes expansion, therefore it can easily accept the reactive site loop of a second alpha1-AT molecule and consequently form polymers of alpha1-AT. These polymers of alpha1-AT aggregate in the hepatocytes and show liver and lungs diseases. Contrary to this, the S variant of alpha1-AT is not associated with any significant clinical disease because the conformation of the inhibitor is not altered significantly. The Z related pathologies could be treated by liver transplantation, augmentation therapy, gene therapy, peptide therapy and chemical chaperone therapy. In addition to common deficiency variants, there are several rare deficiency variants of alpha1-AT like Siiyama, Mmalton, Mprocida, Mheerlen, Mmineral springs, Mnichinan, Pduarte, Wbethesda Zaugsberg, and Zbristol. In Siiyama, Mmalton, Mnichinan and Zaugsberg, the beta-sheet A is present in an open state therefore these variants readily undergo polymerization and consequently show aggregation in the hepatocytes. In Mprocida, Mheerlen, Mmineral springs, Pduarte and Wbethesda the conformation is altered significantly therefore these variants become conformationally less stable and thereby undergo intracellular proteolysis. These rare genetic variants show lungs and / or liver disease. There are several null variants of alpha1-AT that are not detected either at the stage of transcription or translation. The examples of some of the null variants are QOcardiff, QOhong kong, QOgranite falls, QObellingham, QOmattawa, QObolton, and QOludwigshafen. The molecular basis of deficiency of these variants also forms the theme of this review.

Identification of mutations at the antigenic and glycosylation sites in hemagglutinin protein of H5N1 strain.

Hemagglutinin (HA) is the principal antigen, present on the viral surface. It is the primary target for neutralizing antibodies. In this paper, we have carried out studies on human hemagglutinin protein from H5N1 strain with homologous hemagglutinin from non-human sources of H5N1 strains. In all strains, part of the antigenic site (128-141) predicted by computer program "Antigenic", corresponds to immunodominant site Sa of H1 subtype. In AAF02304 strain, A156-->S156 mutation lies at the antigenic subsite of site 2 that corresponds to site B in the H3 subtype. In some strains of non-human origins, there are mutations at the antigenic sites. Interestingly, in AAY56367 strain mutation L138-->H138 lies at the receptor binding site, which also overlaps the antigenic site. Therefore, this amino acid substitution may influence both the specificity of receptor recognition and antibody binding. Seven potential glycosylation sites in human HA and in some strains of non-human sources have been predicted by computer program, Scan Prosite. In some strains of HA from non-human sources because of mutation, an additional glycosylation site appeared at the antigenic site. Therefore in these strains the oligosaccharides will mask the surface of HA as well as antigenic site. Hence these strains will not be recognized by host immune system.

Studies on the acid induced unfolding of human serum albumin.

The acid induced unfolding of HSA (Human Serum Albumin) was studied using UV-difference spectroscopy, fluorescence spectroscopy and far-UV CD spectroscopy. In UV-difference spectroscopy, the molar extinction coefficient decreased from N state to F state. Partially buried Tyr residues are transferred from a medium of high polarizability (native N state) to a medium of low polarizability (F state). This is followed by loss of two electrostatic interactions Lys 205-Glu 465 and Arg218-Asp451 or one electrostatic interaction Lys205-Glu 465/Arg218-Asp451 and one buried carboxyl group of acidic amino acid. Similarly, UV-difference spectroscopy showed a decrease in absorbance in F<-->E transition due to exposure of completely buried tyrosine residues from medium of high polarizability (F state) to a medium of low polarizability (E state). This is also followed by loss of one electrostatic interaction out of three electrostatic interactions namely, Asp187-Lys432, Asp187-Lys521 and Lys190-Glu425. The tryptophanyl fluorescence spectra showed that the N<-->F transition is accompanied by a decrease in fluorescence intensity. This implies that there is partial exposure of Trp214 to aqueous environment. Consequently, there is a loss of two electrostatic interactions Lys 205-Glu 465 and Arg218-Asp451 or one electrostatic interaction Lys205-Glu 465/Arg218-Asp451 and one buried carboxyl group of acidic amino acid in N<-->F transition. The tryptophanyl fluorescence spectroscopy also showed that partially exposed Trp214 residue becomes nearly completely exposed in F<-->E transition. This is also followed by loss of two electrostatic interactions out of three Asp 187-Lys432,Asp187-Arg521 and Lys190-Glu425 in F<-->E transition. Taken together, these results showed that in N<-->F and F<-->E transitions a different number of electrostatic interaction is detected by different techniques. Secondly, in both N<-->F and F<-->E transitions, chromophoric groups are exposed first to aqueous environment and this is followed by loss of electrostatic interactions.

Urea and acid induced unfolding of fatted and defatted human serum albumin.

Urea induced equilibrium unfolding of fatted and defatted human serum albumin (HSA) showed that fatty acid stabilizes native and intermediate states. Similarly acid induced unfolding of fatted and defatted HSA also showed that fatty acid stabilizes Ntwo left arrowsF and Ftwo left arrowsE transitions. These results also showed that five electrostatic interactions along with one buried carboxyl group of acidic amino acid are involved in acid induced unfolding of HSA.