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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.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59–127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart., Peterle et al. show that a subtilisin like serine protease secreted from gut microbiota Bacillus subtilis cleaves the wild-type human transthyretin (hTTR) to generate an amyloidogenic peptide. High propensity of the hTTR fragment to form pathogenic protein aggregates implicates the serine protease in the pathogenesis of acquired senile systemic amyloidosis.

Peptides are attractive drugs because of their specificity and minimal off-target effects. Short half-lives are within their major drawbacks, limiting actual use in clinics. The golden standard in therapeutic peptide development implies identification of a minimal core sequence, then modified to increase stability through several strategies, including the introduction of nonnatural amino acids, cyclization, and lipidation. Here, we investigated plasma degradations of hormone sequences all composed of a minimal active core peptide and a C-terminal extension. We first investigated pro-opimelanocortin (POMC) γ2/γ3-MSH hormone behavior and extended our analysis to POMC-derived α-melanocyte stimulating hormone/adrenocorticotropic hormone signaling neuropeptides and neurotensin. We demonstrated that in all the three cases analyzed in this study, few additional residues mimicking the natural sequence alter both peptide stability and the mechanism(s) of degradation of the minimal conserved functional pattern. Our results suggest that the impact of extensions on the bioactivity of a peptide drug has to be carefully evaluated throughout the optimization process.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly people. To date, drugs able to reverse the disease are not available; the gold standard is levodopa that only relieves clinical symptoms, yet with severe side effects after prolonged administration. Many efforts are underway to find alternative targets for PD prevention or treatment, the most promising being α-synuclein (Syn). Recently, we reported that oleuropein aglycone (OleA) interferes with amyloid aggregation of Syn both stabilizing its monomeric state and inducing the formation of harmless, off-pathway oligomers. This study is focused at describing the interaction between Syn and hydroxytyrosol (HT), the phenolic moiety and main metabolite of OleA, and the interferences with Syn aggregation by using biophysical and biological techniques. Our results show that HT dose-dependently inhibits Syn aggregation and that covalent and non-covalent binding mediate HT-Syn interaction. HT does not modify the natively unfolded structure of Syn, rather, it stabilizes specific regions of the molecule leading to inhibition of protein fibrillation. Cellular assays showed that HT reduces the toxicity of Syn aggregates. Moreover, Syn aggregates interaction with the cell membrane, an important factor for prion-like properties of Syn on-pathway oligomers, was reduced in cells exposed to Syn aggregates grown in the presence of HT.

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.