Recent discoveries on the self-assembly of aromatic amino acids into amyloid-like neurotoxic nanostructures have initiated a quest to decode the molecular mechanisms for the initiation of neurodegeneration. Moreover, the multicomponent nature of the amyloid deposits still questions the existing and well-defined amyloid cascade hypothesis. Hence, deciphering the neurotoxicity of amyloid-like nanostructures of aromatic amino acids becomes crucial for understanding the etiology of amyloidogenesis. Here, we demonstrate the cellular internalization and consequential damaging effects of self-assembled amyloid-like tryptophan nanostructures on human neuroblastoma cells. The cell-damaging potential of tryptophan nanostructure seems to be facilitated via ROS generation, necrosis and apoptosis mediated cell death. Further, tryptophan nanostructures were found to be seeding competent conformers, which triggered aggressive aggregation of brain extract components. The early stage intermediate nanostructures possess a higher cross-seeding efficacy than the seeding potential of the matured tryptophan fibrils. In addition to the cell-damaging and cross-seeding effects, tryptophan fibrils were found to catalyze oxidation of neuromodulator dopamine. These findings add more insights into the specific role of tryptophan self-assembly during the pathogenesis of hypertryptophanemia and other amyloid-associated neurodegenerative complications.

Tryptophan is an essential amino acid, which is not only a building block for protein synthesis, but also a precursor for the biosynthesis of co-enzymes and neuromodulators, such as NAD/NADP(H), kynurenic acid, melatonin and serotonin. It also plays a role in immune homeostasis, as local tryptophan catabolism impairs T-lymphocyte mediated immunity. Therefore, tryptophan plasmatic concentration needs to be stable, in spite of large variations in dietary supply. Here, we review the main checkpoints accounting for tryptophan homeostasis, including absorption, transport, metabolism and elimination, and we discuss the physiopathology of disorders associated with their dysfunction. Tryptophan is catabolized along the kynurenine pathway through the action of two enzymes that mediate the first and rate-limiting step of the pathway: indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO). While IDO1 expression is restricted to peripheral sites of immune modulation, TDO is massively expressed in the liver and accounts for 90% of tryptophan catabolism. Recent data indicated that the stability of the TDO protein is regulated by tryptophan and that this regulation allows a tight control of tryptophanemia. TDO is stabilized when tryptophan is abundant in the plasma, resulting in rapid degradation of dietary tryptophan. In contrast, when tryptophan is scarce, TDO is degraded by the proteasome to avoid excessive tryptophan catabolism. This is triggered by the unmasking of a degron in a non-catalytic tryptophan-binding site, resulting in TDO ubiquitination by E3 ligase SKP1-CUL1-F-box. Deficiency in TDO or in the hepatic aromatic transporter SLC16A10 leads to severe hypertryptophanemia, which can disturb immune and neurological homeostasis.

Dietary consumption of Trp via protein-based foods is essential for the maintenance of crucial metabolic processes including the synthesis of proteins and several vital metabolites such as serotonin, melatonin, acetyl CoA, and NADP. However, the abnormal build-up of Trp is known to cause familial hypertryptophanemia and several brain-related medical complications. The molecular mechanism of the onset of such Trp-driven health issues is largely unknown. Here, we show that Trp, under the physiologically mimicked conditions of temperature and buffer, undergoes a concentration driven self-assembly process, yielding amyloid-mimicking nanofibers. Viable H-bonds, π-π interactions and hydrophobic contacts between optimally coordinated Trp molecules become important factors for the formation of a Trp nanoassembly that displays a hydrophobic exterior and a hydrophilic interior. Importantly, Trp nanofibers were found to possess high affinity for native proteins, and they act as cross-seeding competent conformers capable of nucleating amyloid formation in globular proteins including whey protein β-lactoglobulin and type II diabetes linked insulin hormone. Moreover, these amyloid mimicking Trp nanostructures showed toxic effects on neuroblastoma cells. Since the key symptoms in hypertryptophanemia such as behavioural defects and brain-damaging oxidative stress are also observed in amyloid related disorders, our findings on amyloid-like Trp-nanofibers may help in the mechanistic understanding of Trp-related complications and these findings are equally important for innovation in applied nanomaterials design and strategies.

There is a plethora of significant research that illustrates toxic self-assemblies formed by the aggregation of single amino acids, such as phenylalanine, tyrosine, tryptophan, cysteine, and methionine, and their implication on the etiology of inborn errors of metabolisms (IEMs), such as phenylketonuria, tyrosinemia, hypertryptophanemia, cystinuria, and hypermethioninemia, respectively. Hence, studying the aggregation behavior of single amino acids is very crucial from the chemical neuroscience perspective to understanding the common etiology between single amino acid metabolite disorders and amyloid diseases like Alzheimer's and Parkinson's. Herein we report the aggregation properties of nonaromatic single amino acids l-proline (Pro), l-hydroxyproline (Hyp), and l-lysine hydrochloride (Lys). The morphologies of the self-assembled structures formed by Pro, Hyp, and Lys were extensively studied by various microscopic techniques, and controlled morphological transitions were observed under varied concentrations and aging times. The mechanism of structure formation was deciphered by concentration-dependent 1H NMR analysis, which revealed the crucial role of hydrogen bonding and hydrophobic interactions in the structure formation of Pro, Hyp, and Lys. MTT assays on neural (SHSY5Y) cell lines revealed that aggregates formed by Pro, Hyp, and Lys reduced cell viability in a dose-dependent manner. These results may have important implications in the understanding of the patho-physiology of disorders such as hyperprolinemia, hyperhydroxyprolinemia, and hyperlysinemia since all these IEMs are associated with severe neurodegenerative symptoms, including intellectual disability, seizures, and psychiatric problems. Our future studies will endeavor to study these biomolecular assemblies in greater detail by immuno-histochemical analysis and advanced biophysical assays.

Systemic mastocytosis (SM) is characterized by a clonal proliferation of neoplastic mast cells (MCs) in one or more extracutaneous organs including the bone marrow (BM). SM is often associated with osteoporosis (OP) and fractures. Hypertryptasemia usually occurs in SM. We investigated the prevalence of hypertryptasemia in a series of severe osteoporotic patients, the performance of the tryptase test in diagnosing SM in these patients, and their bone features. The medical records of 232 patients (168 females and 64 males) with a diagnosis of OP (50.4% with fractures) and a serum tryptase assessment were reviewed. BM assessment was performed in a subset of hypertryptasemic patients; clinical, biochemical, and radiographic data were collected. Hypertryptasemia was detected in 33 patients. BM assessment (n = 16) was normal in 8 hypertryptasemic patients, while BM criteria for the diagnosis of SM were met in 3 patients, MC alterations were detected in 4 patients, and one patient presented a polycythemia vera. Serum tryptase levels were higher than 11.4 ng/ml in all patients with BM alterations. The best cut-off of tryptase level related to BM alterations was 17.9 ng/ml, with a sensibility and sensitivity of 75% (AUC = 0.797 and P = 0.015 by ROC analysis). All osteoporotic patients with hypertryptasemia experienced at least one vertebral fracture associated with a severe reduction of the lumbar bone mineral density. The prevalence of MC-related disorders in severe OP was 3.0%, accounting for the 7.4% of the secondary causes of OP. MC-related disorders may be involved in bone fragility and assessment of serum tryptase is useful to detect MC-related disorders.