Structure-function relationship of new crotamine isoform from the Crotalus durissus cascavella

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7-3.0-fold than to control. The Na+ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na+ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic beta-cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

A novel supersecondary structure in globular proteins comprising the collagen-like helix and ?-turn

A structure consisting of the polyproline-II or collagen-like helix immediately succeeded by a ?-turn is seen in several synthetic peptides and has been suggested to be the conformational requirement for proline hydroxylation in nascent procollagen. Using a simple algorithm for detecting secondary structures, we have analysed crystal structure data on 40 globular proteins and have found eight examples of the collagen-helix + ?-turn supersecondary structure in 15 proteins that contain the collagen-like helical segments.

Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

Review: formation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins.

Synucleins are small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. alpha-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of alpha-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of alpha-synuclein has also been identified as the non-Abeta component of Alzheimer's Disease amyloid. In this review we describe some structural properties of alpha-synuclein and the two mutant forms, as well as alpha-synuclein fragments, with particular emphasis on their ability to form beta-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by alpha-synuclein and the two mutant proteins are highlighted. Interactions between alpha-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of alpha-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.