Insights into amyloid fibril formation from mass spectrometry

Mass spectrometry has become increasingly important in amyloid research specifically in the mechanism of formation and characterization of fibrils. In this review we highlight key experiments that provide evidence for different conformations, interactions and unfolding intermediates in proteins associated with amyloid diseases.     

Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer’s disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2–Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer’s disease.     

Short peptide amyloid organization: stabilities and conformations of the islet amyloid peptide NFGAIL

Experimentally, short peptides have been shown to form amyloids similar to those of their parent proteins. Consequently, they present useful systems for studies of amyloid conformation. Here we simulate extensively the NFGAIL peptide, derived from the human islet amyloid polypeptide (residues 22-27). We simulate different possible strand/sheet organizations, from dimers to nonamers. Our simulations indicate that the most stable conformation is an antiparallel strand orientation within the sheets and parallel between sheets. Consistent with the alanine mutagenesis, we find that the driving force is the hydrophobic effect. Whereas the NFGAIL forms stable oligomers, the NAGAIL oligomer is unstable, and disintegrates very quickly after the beginning of the simulation. The simulations further identify a minimal seed size. Combined with our previous simulations of the prion-derived AGAAAAGA peptide, AAAAAAAA, and the Alzheimer Abeta fragments 16-22, 24-36, 16-35, and 10-35, and the solid-state NMR data for Abeta fragments 16-22, 10-35, and 1-40, some insight into the length and the sequence matching effects may be obtained.     

Insights from protein-protein interaction studies on bacterial pathogenesis

Introduction: The threat bacterial pathogens pose to human health is increasing with the number and distribution of antibiotic-resistant bacteria, while the rate of discovery of new antimicrobials dwindles. Proteomics is playing key roles in understanding the molecular mechanisms of bacterial pathogenesis, and in identifying disease outcome determinants. The physical associations identified by proteomics can provide the means to develop pathogen-specific treatment methods that reduce the spread of antibiotic resistance and alleviate the negative effects of broad-spectrum antibiotics on beneficial bacteria.

Areas covered: This review discusses recent trends in proteomics and introduces new and developing approaches that can be applied to the study of protein-protein interactions (PPIs) underlying bacterial pathogenesis. The approaches examined encompass options for mapping proteomes as well as stable and transient interactions in vivo and in vitro. We also explored the coverage of bacterial and human-bacterial PPIs, knowledge gaps in this area, and how they can be filled.

Expert commentary: Identifying potential antimicrobial candidates is confounded by the complex molecular biology of bacterial pathogenesis and the lack of knowledge about PPIs underlying this process. Proteomics approaches can offer new perspectives for mechanistic insights and identify essential targets for guiding the discovery of next generation antimicrobials.     

Insights into protein biosynthesis from structures of bacterial ribosomes

Understanding the structural basis of protein biosynthesis on the ribosome remains a challenging problem for cryo-electron microscopy and X-ray crystallography. Recent high-resolution structures of the Escherichia coli 70S ribosome without ligands, and of the Thermus thermophilus and E. coli 70S ribosomes with bound mRNA and tRNAs, reveal many new features of ribosome dynamics and ribosome-ligand interactions. In addition, the first high-resolution structures of the L7/L12 stalk of the ribosome, responsible for translation factor binding and GTPase activation, reveal the structural basis of the high degree of flexibility in this region of the ribosome. These structures provide groundbreaking insights into the mechanism of protein synthesis at the level of ribosome architecture, ligand binding and ribosome dynamics.     

Insights from genomic comparisons of genetically monomorphic bacterial pathogens

Some of the most deadly bacterial diseases, including leprosy, anthrax and plague, are caused by bacterial lineages with extremely low levels of genetic diversity, the so-called 'genetically monomorphic bacteria'. It has only become possible to analyse the population genetics of such bacteria since the recent advent of high-throughput comparative genomics. The genomes of genetically monomorphic lineages contain very few polymorphic sites, which often reflect unambiguous clonal genealogies. Some genetically monomorphic lineages have evolved in the last decades, e.g. antibiotic-resistant Staphylococcus aureus, whereas others have evolved over several millennia, e.g. the cause of plague, Yersinia pestis. Based on recent results, it is now possible to reconstruct the sources and the history of pandemic waves of plague by a combined analysis of phylogeographic signals in Y. pestis plus polymorphisms found in ancient DNA. Different from historical accounts based exclusively on human disease, Y. pestis evolved in China, or the vicinity, and has spread globally on multiple occasions. These routes of transmission can be reconstructed from the genealogy, most precisely for the most recent pandemic that was spread from Hong Kong in multiple independent waves in 1894.     

Insights from modeling three-dimensional structures of the human potassium and sodium channels

Ion channels allow the movement of ions across cell membranes. Nearly all cells have membranes spanned by ion channels, without which human nerves simply would not work. Ion channels are formed by the aggregation of subunits into a cylindrical configuration that allows a pore, thus forming a kind of tube for ion trafficking. In the present study, the subunits of the human potassium channel are formed by four identical protein chains, whereas for the case of the human sodium channel, the corresponding subunits are actually four hetero-domains formed by the folding of a very large but single protein chain. Since both of the two ion channels are important targets for drug discovery, the 3D (dimensional) structures of their pore regions were developed. On the basis of the 3D models, some important molecular biological mechanisms were discussed that may stimulate novel strategies for therapeutic treatment of the diseases related to ion channel disorders, such as long QT syndrome and chronic pain.     

A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor

The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.     

Helix-dipole effects in peptide self-assembly to amyloid

The formation of amyloid fibrils is associated with incurable diseases including Alzheimer's, Parkinson's, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown partly because of the absence of a clear structural characterization of intermediates. There is experimental evidence, however, for α-helical intermediates that has come primarily from circular dichroism spectroscopy. Here, we strengthen the evidence for helical intermediates by demonstrating helix-dipole effects in the early events of self-assembly. Previously, we showed that capped peptides containing the part of the islet amyloid polypeptide that may be responsible for the initial intermolecular contacts (Acetyl-R(11)LANFLVHSSNNFGA(25)-NH(2) and Acetyl-R(11)LANFLVHSGNNFGA(25)-NH(2) which contains the S20G mutation associated with early onset type 2 diabetes) self-assemble via helical intermediates [Liu et al. (2010) J. Am. Chem. Soc.132, 18223-18232]. We demonstrate here that when the peptides are uncapped, they do not self-assemble as indicated primarily by circular dichroism and nuclear magnetic resonance data. Self-assembly is restored when the charge on α-NH(3)(+) of Arg11 is eliminated but not when the charge on α-COO(-) of Ala25 is removed, consistent with the helicity of the peptides skewed toward the N-terminus. Our results strengthen the hypothesis that α-helical intermediates are on pathway to amyloid formation and indicate that the helix dipole is an attractive target for inhibiting the formation of α-helical assemblies.     

A novel peptide from amyloid P component supports cell attachment.

Amyloid P component is a glycoprotein found in association with connective tissues throughout the body and is also a component of human serum. We have identified a dodecapeptide from amyloid P component which is capable of supporting the attachment of a wide variety of cells to the surface of polystyrene plastic dishes. 83% of the activity is confined to a hexapeptide, FTLCFR. Saturation of cell attachment occurs at a peptide concentration of 100 micrograms/ml used to coat the plastic. These results indicate that the active peptide may represent a functional property of amyloid P component which heretofore has no function.     

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.     

Insights into bacterial protein glycosylation in human microbiota

The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.     

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain

Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses. Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding.     

How to quantify protein diffusion in the bacterial membrane

Lateral diffusion of proteins in the plane of a biological membrane is important for many vital processes, including energy conversion, signaling, chemotaxis, cell division, protein insertion, and secretion. In bacteria, all these functions are located in a single membrane. Therefore, quantitative measurements of protein diffusion in bacterial membranes can provide insight into many important processes. Diffusion of membrane proteins in eukaryotes has been studied in detail using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single-molecule fluorescence (SMF) microscopy. In case of bacteria, such experiments are intrinsically difficult due to the small size of the cells. Here, we review these experimental approaches to quantify diffusion in general and their strengths and weaknesses when applied to bacteria. In addition, we propose a method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs). We demonstrate the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins. Using computer simulations, we study the effect of cell dimensions and membrane curvature on measured CPDs. We find that at least two mobile populations with distinct diffusion coefficients (of 7 and 169 nm(2) ms(-1) , respectively) are necessary to explain the experimental data. The approach described here should be widely applicable for the quantification of membrane-protein diffusion in living bacteria.