Compound heterozygosis in AADC deficiency: A complex phenotype dissected through comparison among heterodimeric and homodimeric AADC proteins.

Compound heterozygosis is the most diffuse and hardly to tackle condition in aromatic amino acid decarboxylase (AADC) deficiency, a genetic disease leading to severe neurological impairment. Here, by using an appropriate vector, we succeeded in obtaining high yields of AADC protein and characterizing two new heterodimers, T69M/S147R and C281W/M362T, detected in two AADC deficiency patients. We performed an extensive biochemical characterization of the heterodimeric recombinant proteins and of the related homodimers, by a combination of dichroic and fluorescence spectroscopy and activity assays together with bioinformatic analyses. We found that T69M/S147R exhibits negative complementation in terms of activity but it is more stable than the average of the homodimeric counterparts. The heterodimer C281W/M362T retains a nearly good catalytic efficiency, whereas M362T homodimer is less affected and C281W homodimer is recovered as insoluble. These results, which are consistent with the related phenotypes, and the data emerging from previous studies, suggest that the severity of AADC deficiency is not directly explained by positive or negative complementation phenomena, but rather depends on: i) the integrity of one or both active sites; ii) the structural and functional properties of the entire pool of AADC proteins expressed. Overall, this integrated and cross-sectional approach enables proper characterization and depicts the functional result of subunit interactions in the dimeric structure and will help to elucidate the physio-pathological mechanisms in AADC deficiency.

α-Synuclein structural features inhibit harmful polyunsaturated fatty acid oxidation, suggesting roles in neuroprotection.

α-Synuclein (aS) is a protein abundant in presynaptic nerve terminals in Parkinson disease (PD) and is a major component of intracellular Lewy bodies, the pathological hallmark of neurodegenerative disorders such as PD. Accordingly, the relationships between aS structure, its interaction with lipids, and its involvement in neurodegeneration have attracted great interest. Previously, we reported on the interaction of aS with brain polyunsaturated fatty acids, in particular docosahexaenoic acid (DHA). aS acquires an α-helical secondary structure in the presence of DHA and, in turn, affects DHA structural and aggregative properties. Moreover, aS forms a covalent adduct with DHA. Here, we provide evidence that His-50 is the main site of this covalent modification. To better understand the role of His-50, we analyzed the effect of DHA on aS-derived species: a naturally occurring variant, H50Q; an oxidized aS in which all methionines are sulfoxides (aS4ox); a fully lysine-alkylated aS (acetyl-aS); and aS fibrils, testing their ability to be chemically modified by DHA. We show, by mass spectrometry and spectroscopic techniques, that H50Q and aS4ox are modified by DHA, whereas acetyl-aS is not. We correlated this modification with aS structural features, and we suggest a possible functional role of aS in sequestering the early peroxidation products of fatty acids, thereby reducing the level of highly reactive lipid species. Finally, we show that fibrillar aS loses almost 80% of its scavenging activity, thus lacking a potentially protective function. Our findings linking aS scavenging activity with brain lipid composition suggest a possible etiological mechanism in some neurodegenerative disorders.

α-Synuclein and Polyunsaturated Fatty Acids: Molecular Basis of the Interaction and Implication in Neurodegeneration.

α-Synuclein (α-syn) is a 140-amino acid protein, the physiological function of which has yet to be clarified. It is involved in several neurodegenerative disorders, and the interaction of the protein with brain lipids plays an important role in the pathogenesis of Parkinson's disease (PD). Polyunsaturated fatty acids (PUFA) are highly abundant in the brain where they play critical roles in neuronal membrane fluidity and permeability, serve as energy reserves and function as second messengers in cell signaling. PUFA concentration and composition in the brain are altered with age when also an increase of lipid peroxidation is observed. Considering that PD is clearly correlated with oxidative stress, PUFA abundance and composition became of great interest in neurodegeneration studies because of PUFA's high propensity to oxidize. The high levels of the PUFA docosahexaenoic acid (DHA) in brain areas containing α-syn inclusions in patients with PD further support the hypothesis of possible interactions between α-syn and DHA. Additionally, a possible functional role of α-syn in sequestering the early peroxidation products of fatty acids was recently proposed. Here, we provide an overview of the current knowledge regarding the molecular interactions between α-syn and fatty acids and the effect exerted by the protein on their oxidative state. We highlight recent findings supporting a neuroprotective role of the protein, linking α-syn, altered lipid composition in neurodegenerative disorders and PD development.

Zymogen activation and subcellular activity of subtilisin kexin isozyme 1/site 1 protease.

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B'/B followed by the herein newly identified C'/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B'/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1-P8) and P1' are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates.

Light exacerbates local and global effects induced by pH unfolding of Ipilimumab.

Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage, and administration processes, these proteins encounter various stressors such as temperature fluctuations, vibrations, and light exposure, able to induce chemico-physical modifications to their structure. Viral inactivation is a key step in downstream processes, and it is achieved by titration of the mAb at low pH, followed by neutralization. The changes of the pH pose a significant risk of unfolding and subsequent aggregation to proteins, thereby affecting their manufacturing. This study aims to investigate whether a combined exposure to light during the viral inactivation process can further affect the structural integrity of Ipilimumab, a mAb primarily used in the treatment of metastatic melanoma. The biophysical and biochemical characterization of Ipilimumab revealed that pH variation is a considerable risk for its stability with irreversible unfolding at pH 2. The threshold for Ipilimumab denaturation lies between pH 2 and 3 and is correlated with the loss of the protein structural cooperativity, which is the most critical factor determining the protein refolding. Light has demonstrated to exacerbate some local and global effects making pH-induced exposed regions more vulnerable to structural and chemical changes. Therefore, specific precautions to real-life exposure to ambient light during the sterilization process of mAbs should be considered to avoid loss of the therapeutic activity and to increase the yield of production. Our findings underscore the critical role of pH optimization in preserving the structural integrity and therapeutic efficacy of mAbs. Moreover, a detailed conformational study on the structural modifications of Ipilimumab may improve the chemico-physical knowledge of this effective drug and suggest new production strategies for more stable products under some kind of stress conditions.

Impact of Post Manufacturing Handling of Protein-Based Biologic Drugs on Product Quality and User Centricity.

This article evaluates the current gaps around the impact of post-manufacturing processes on the product qualities of protein-based biologics, with a focus on user centricity. It includes the evaluation of the regulatory guidance available, describes a collection of scientific literature and case studies to showcase the impact of post-manufacturing stresses on product and dosing solution quality. It also outlines the complexity of clinical handling and the need for communication, and alignment between drug providers, healthcare professionals, users, and patients. Regulatory agencies provide clear expectations for drug manufacturing processes, however, guidance supporting post-product manufacturing handling is less defined and often misaligned. This is problematic as the pharmaceutical products experience numerous stresses and processes which can potentially impact drug quality, safety and efficacy. This article aims to stimulate discussion amongst pharmaceutical developers, health care providers, device manufacturers, and public researchers to improve these processes. Patients and caregivers' awareness can be achieved by providing relevant educational material on pharmaceutical product handling.

Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid.

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.

Dopamine quinones interact with alpha-synuclein to form unstructured adducts.

alpha-Synuclein (alphasyn) fibril formation is considered a central event in the pathogenesis of Parkinson's disease (PD). In recent years, it has been proposed that prefibrillar annular oligomeric beta-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit alphasyn fibril formation supporting the idea that DAQ might stabilize alphasyn protofibrils. In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized alphasyn/DAQ adducts. Contrary to protofibrils, we demonstrated that alphasyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on alphasyn by DAQ. Our results indicate that only a small fraction of alphasyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on alphasyn.

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids.

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b(6)f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b(6)f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.

Amyloid fibril formation and disaggregation of fragment 1-29 of apomyoglobin: insights into the effect of pH on protein fibrillogenesis.

The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb(1-29)) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of beta-sheet structure. ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to 8.3. It is of interest that amyloid-like fibrils form again if the pH of the solution is brought back to 2.0. While apoMb(1-29) fibrils obtained at pH 2.0 are resistant to proteolysis by pepsin, the disaggregated fibrils are easily cleaved at pH 8.3 by trypsin and V8 protease, and some of the resulting fragments aggregate very quickly in the proteolysis mixture, forming amyloid-like fibrils. We show that the increase of amyloidogenicity of apoMb(1-29) following acidification or proteolysis at pH 8.3 can be attributed to the decrease of the peptide net charge following these alterations. The results observed here for apoMb(1-29) provide an experimental basis for explaining the effect of charge and pH on amyloid fibril formation by both unfolded and folded protein systems.

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution.

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of "sequence scrambled" variants of the first 29 residues of horse heart apomyoglobin (apoMb(1-29)), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k(agg)) and a remarkable shortening of the lag phase (t(lag)) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k(agg) and t(lag) values of the apoMb(1-29) variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.

Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability.

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74 degrees C) of native decorsin. The mono-substituted analogs display a Tm of 66-67 degrees C, while the double-substituted analog a Tm of 50 degrees C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro-->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the 'proline rule', previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.

Protein dissection enhances the amyloidogenic properties of alpha-lactalbumin.

Alpha-lactalbumin (LA) in its molten globule (MG) state at low pH forms amyloid fibrils. Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the beta-subdomain of the native protein (1-40/53-123, named desbeta-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. Th1-LA and desbeta-LA aggregate at pH 2.0 much faster than the intact protein and form long and well-ordered fibrils. Furthermore, in contrast to intact LA, the LA derivatives form regular fibrils also at neutral pH, even if at much reduced rate. In acidic solution, Th1-LA and desbeta-LA adopt a MG state which appears to be similar to that of intact LA, as given by spectroscopic criteria. At neutral pH, both Th1-LA and desbeta-LA are able to bind the hydrophobic dye 1-anilinonaphtalene-8-sulfonate, thus indicating the presence of exposed hydrophobic patches. It is concluded that nicked Th1-LA and gapped desbeta-LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein species to allow the large conformational transitions required for the polypeptide chain to form the amyloid cross-beta structure. As a matter of fact, the MG of LA attains an even more flexible conformational state during the early phases of the aggregation process at acidic pH, as deduced from the enhancement of its susceptibility to proteolysis by pepsin. Our data indicate that deletion of the beta-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils, implying that this chain region is not essential for the amyloid formation. It is proposed that a proteolytic hydrolysis of a protein molecule at the cellular level can trigger an easier formation of amyloid precipitates and therefore that limited proteolysis of proteins can be a causative mechanism of protein aggregation and fibrillogenesis. Indeed, a vast majority of protein deposits in amyloid diseases are given by protein fragments derived from larger protein precursors.

The functional dissection of the plasma corona of SiO2-NPs spots histidine rich glycoprotein as a major player able to hamper nanoparticle capture by macrophages.

A coat of strongly-bound host proteins, or hard corona, may influence the biological and pharmacological features of nanotheranostics by altering their cell-interaction selectivity and macrophage clearance. With the goal of identifying specific corona-effectors, we investigated how the capture of amorphous silica nanoparticles (SiO2-NPs; Ø = 26 nm; zeta potential = -18.3 mV) by human lymphocytes, monocytes and macrophages is modulated by the prominent proteins of their plasma corona. LC MS/MS analysis, western blotting and quantitative SDS-PAGE densitometry show that Histidine Rich Glycoprotein (HRG) is the most abundant component of the SiO2-NP hard corona in excess plasma from humans (HP) and mice (MP), together with minor amounts of the homologous Kininogen-1 (Kin-1), while it is remarkably absent in their Foetal Calf Serum (FCS)-derived corona. HRG binds with high affinity to SiO2-NPs (HRG Kd ∼2 nM) and competes with other plasma proteins for the NP surface, so forming a stable and quite homogeneous corona inhibiting nanoparticles binding to the macrophage membrane and their subsequent uptake. Conversely, in the case of lymphocytes and monocytes not only HRG but also several common plasma proteins can interchange in this inhibitory activity. The depletion of HRG and Kin-1 from HP or their plasma exhaustion by increasing NP concentration (>40 µg ml(-1) in 10% HP) lead to a heterogeneous hard corona, mostly formed by fibrinogen (Fibr), HDLs, LDLs, IgGs, Kallikrein and several minor components, allowing nanoparticle binding to macrophages. Consistently, the FCS-derived SiO2-NP hard corona, mainly formed by hemoglobin, α2 macroglobulin and HDLs but lacking HRG, permits nanoparticle uptake by macrophages. Moreover, purified HRG competes with FCS proteins for the NP surface, inhibiting their recruitment in the corona and blocking NP macrophage capture. HRG, the main component of the plasma-derived SiO2-NPs' hard corona, has antiopsonin characteristics and uniquely confers to these particles the ability to evade macrophage capture.

Probing protein structure by limited proteolysis.

Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, alpha-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.

Global analysis of protein structural changes in complex proteomes.

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

Variations of the corona HDL:albumin ratio determine distinct effects of amorphous SiO2 nanoparticles on monocytes and macrophages in serum.

AIM: We investigated monocyte and macrophage death and cytokine production induced by amorphous silica nanoparticles (SiO2-NPs) to clarify the role of defined serum corona proteins. MATERIALS & METHODS: The cytotoxic proinflammatory effects of SiO2-NPs on human monocytes and macrophages were characterized in no serum, in fetal calf serum and in the presence of purified corona proteins. RESULTS: In no serum and in fetal calf serum above approximately 75 µg/ml, SiO2-NPs lysed monocytes and macrophages by plasma membrane damage (necrosis). In fetal calf serum below approximately 75 µg/ml, SiO2-NPs triggered an endolysosomal acidification and caspase-1-dependent monocyte death (pyroptosis). The corona high-density lipoproteins:albumin ratio accounted for the features of the SiO2-NPs in serum. DISCUSSION: Corona high-density lipoproteins are a major determinant of the differential cytotoxic action of SiO2-NPs on monocytes and macrophages.

Comparative proteomic analysis of ductal breast carcinoma demonstrates an altered expression of chaperonins and cytoskeletal proteins.

The aim of the present study was to analyze the protein composition of ductal breast carcinoma and the surrounding normal tissue in individual patients using comparative 2D proteomics and mass spectrometry to detect candidate disease biomarkers for diagnosis and prognosis. Samples of normal and cancerous tissue obtained form 28 patients were analyzed. Chaperonins and cytoskeletal proteins predominated among the 11 proteins for which major changes in abundance were detected. Of these 11 proteins with an altered expression, 2 had a decreased expression and 9 had an increased expression. In addition, the abundance of a few cytokeratins was also altered; however, they were not capable of serving as specific circulatory biomarkers. The proteins which we observed to exhibit an altered expression in infiltrating ductal breast carcinoma may be exploited as novel targets for therapeutic interventions or represent novel diagnostic/prognostic markers for the early detection of aggressive tumors, particularly those with multridrug-resistant phenotypes during the earlier stages of the disease.

Identifying disordered regions in proteins by limited proteolysis.

Limited proteolysis experiments can be successfully used to detect sites of disorder in otherwise folded globular proteins. The approach relies on the fact that the proteolysis of a polypeptide substrate requires its binding in an extended conformation at the protease's active site and thus an enhanced backbone flexibility or local unfolding of the site of proteolytic attack. A striking correlation was found between sites of limited proteolysis and sites of enhanced chain flexibility of the polypeptide chain, this last evaluated by the crystallographically determined B-factor. In numerous cases, it has been shown that limited proteolysis occurs at chain regions characterized by missing electron density and thus being disordered. Therefore, limited proteolysis is a simple and reliable experimental technique that can detect sites of disorder in proteins, thus complementing the results that can be obtained by the use of other physicochemical and computational approaches.

Covalent α-synuclein dimers: chemico-physical and aggregation properties.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1-104 and 29-140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.