Protein and Peptide Letters - Volume 13, Issue 3, 2006

Protein aggregation has long been experienced as an important problem in the biotechnology industry. It was more recently suggested that a range of disorders including amyloid diseases such as Alzheimer's and Parkinson's diseases and type II diabetes, as well as some forms of cancer are associated with protein misfolding and aggregation. Today, it is generally accepted that aberrant protein aggregation results from the failure of proteins to fold or to remain folded in their native state. The fact that protein aggregation plays a prominent role in diseases that are of increasing importance in the context of presentday human health and welfare has stimulated many investigators to focus their work on this process. Defining the kinetic and thermodynamic properties of the aggregation process and characterising at an atomic level the structures of the various species involved in the formation of amyloid fibrils may indeed suggest strategies to prevent or alleviate amyloidoses. These tasks, however, are technically extremely challenging for several reasons. First, the aggregation process is generally irreversible and thereby studies of its kinetic and thermodynamic behaviour are greatly complicated. Second, a suspension of particles scatters the incident light, which generally imposes serious limitations to the use of optical spectroscopy in structural studies of protein aggregates, although fluctuations in the intensity of light scattering over time may under some conditions provide important information on particle size and shape. Finally, the process of protein aggregation and amyloid formation is thought to follow a hierarchical path of assembly involving multiple steps of association and a variety of conformational rearrangements. The heterogeneity and the transient or insoluble nature of the various species seriously limit the applicability of the two most powerful methods of structural biology, namely solution NMR spectroscopy and X-ray diffraction. As it will become evident from the series of review articles included in this special issue of Protein and Peptide Letters, technical innovations in molecular biology and biophysics have led to a recent blossoming of research devoted to aggregation and amyloid fibril formation, despite all the challenges outlined above. It would clearly be beyond the scope of an issue of this size to give a comprehensive coverage of all techniques that are currently used in this growing field of research. We therefore chose to concentrate on a set of techniques that, in combination with each other, can provide a detailed picture of both kinetic and structural events in protein aggregation and amyloid fibril formation. Each article focuses on a particular technique starting with a general introduction on methodological principles, followed by selected examples that illustrate how it is applied to study mechanistic aspects of peptide and protein assembly. The scope of the first two reviews is a general overview of the field. Dumoulin and Bader summarize some key discoveries in amyloid research ever since Virchow coined the term "amyloid", underlying the technical developments that made them possible. Dobson provides a more general overview into protein folding and misfolding and its link to human disease. He reviews our present knowledge of the nature of these fibrillar aggregates and the manner in which they form, and discusses their origins and potential means of suppressing of the pathogenic properties with which amyloid fibrils and their precursors are associated..............

One of the hallmarks of modern science is technically controlled experimentation. In this paper, we underline how technical developments over the last 150 years have repeatedly created new horizons in amyloid research. The main focus is on chemical and biophysical analyses of amyloid fibrils in vivo and in vitro. Investigations into mechanistic aspects of fibril formation and possible links with pathogenesis are also discussed.

Protein molecules have emerged through evolution so that they are able to remain in their functional and soluble states under normal physiological conditions, although in other situations they often have a high propensity to aggregate. Aggregation in vivo is associated with a wide range of human disorders, including Alzheimer's disease and type II diabetes, medical conditions that are becoming increasingly common in the modern world. In such diseases, aggregated proteins can often be observed as highly intractable thread-like species known as amyloid fibrils. This article provides an overview of our present knowledge of the nature of these fibrillar aggregates and the manner in which they form, and discusses the origins and potential means of suppression of the pathogenic properties with which they and their precursors are associated.

Solid state nuclear magnetic resonance (NMR) has developed into one of the most informative and direct experimental approaches to the characterization of the molecular structures of amyloid fibrils, including those associated with Alzheimer's disease. In this article, essential aspects of solid state NMR methods are described briefly and results obtained to date regarding the supramolecular organization of amyloid fibrils and the conformations of peptides within amyloid fibrils are reviewed.

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer's diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid β-protein.

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.

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.

A common mechanism of conformational changes and pathological aggregation of proteins associated with amyloid diseases remains to be proven. High pressure is emerging as a new strategy for studying aspects of amyloid formation. Pressure provides a convenient means to populate and characterize partially folded states, which are thought to have a key role in assembly processes of proteins into amyloid fibrils. High pressure can also be used to dissociate aggregates and amyloid fibrils or on the opposite to generate such species.

Elucidation of the molecular determinants that drive proteins to aggregate is important both to advance our fundamental understanding of protein folding and misfolding, and as a step towards successful intervention in human disease. Combinatorial strategies enable unbiased and model-free approaches to probe sequence/structure relationships. Through the use of combinatorial methods, it is possible (i) to probe the sequence determinants of natural amyloid proteins by screening libraries of amino acid substitutions (mutations) to identify those that prevent amyloid formation; and (ii) to test new hypotheses about the mechanism of formation of amyloid fibrils by using these hypotheses to guide the design of combinatorial libraries of de novo amyloid-like proteins. Here, we review how these two approaches have been used to study the molecular determinants of protein aggregation and amyloidogenicity.

The process of protein misfolding and aggregation has been associated with an increasing number of pathological conditions that include Alzheimer's and Parkinson's diseases, and type II diabetes. In addition, the discovery that proteins unrelated to any known disorder can be converted into aggregates of morphologies similar to those found in diseased tissue has lead to the recognition that this type of assemblies represents a generic state of polypeptide chains. Therefore, despite the enormous complexity of the in vivo mechanisms that have evolved in living organisms to prevent and control the formation of protein aggregates, the process of aggregation itself appears ultimately to be caused by intrinsic properties of polypeptide chains, in particular by the tendency of the backbone to form hydrogen bonds, and be modulated by the presence of specific patterns of hydrophobic and charged residues. Theoreticians have just recently started to respond to the challenge of identifying the determinants of the aggregation process. In this review, we provide an account of the theoretical results obtained so far.

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by 1H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17- residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.

Functional S100P requires dimer formation and dimerization might form for one of the two reasons: i. producing a pair of site for target protein binding or ii. modulation of cation binding affinity. The extent of exposed protein hydrophobicity was related to dimer formation.

Interaction between S100P and its target protein is an essential step in several cellular functions. The amphiphatic mellitin peptide binds tightly to S100P protein in the presence of calcium cation. Since little is known about the recognition sequence, mellitin interaction form a model for S100P. Interaction between mellitin and protein examined to identify key regions required for the protein-protein interaction.

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the α (30.7 kDa) and β (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45→Arg and Lys56→Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.

Mabinlin II is a thermostable sweet protein isolated from the mature seeds of Capparis masaikai Levl. grown in the subtropical region of the South of China. The Mabinlin II has been crystallized and diffraction data were collected to 1.7 Å resolution. The crystal belongs to space group C2 with unit cell parameters a = 80.11 Å, β = 51.08 Å, c = 47.34 Å, b = 122.77° .