RESUMO
The emergence of a crystal nucleus from disordered states is a critical and challenging aspect of the crystallization process, primarily due to the extremely short length and timescales involved. Methods such as liquid-cell or low-dose focal-series transmission electron microscopy (TEM) are often employed to probe these events. In this study, we demonstrate that ion mobility spectrometry-mass spectrometry (IMS-MS) offers a complementary and insightful perspective on the nucleation process by examining the sizes and shapes of small clusters, specifically those ranging from n = 2 to 40. Our findings reveal the significant role of sulfate ions in the growth of adeninediium sulfate clusters, which are the precursors to the formation of single crystals. Specifically, sulfate ions stabilize adenine clusters at the 1:1 ratio. In contrast, guanine sulfate forms smaller clusters with varied ratios, which become stable as they approach the 1:2 ratio. The nucleation size is predicted to be between n = 8 and 14, correlating well with the unit cell dimensions of adenine crystals. This correlation suggests that IMS-MS can identify critical nucleation sizes and provide valuable structural information consistent with established crystallographic data. We also discuss the strengths and limitations of IMS-MS in this context. IMS-MS offers rapid and robust experimental protocols, making it a valuable tool for studying the effects of various additives on the assembly of small molecules. Additionally, it aids in elucidating nucleation processes and the growth of different crystal polymorphs.
RESUMO
The self-association of metabolites into well-ordered assemblies at the nanoscale has significant biological and medical implications. The thiol-containing amino acid cysteine (CYS) can assemble into amyloid-like nanofibrils, and its oxidized form, the disulfide-bonded cystine (CTE), forms hexagonal crystals as those found in cystinuria due to metabolic disorder. Yet, there have been no attempts to connect these two phenomena, especially the fibril-to-crystal transition. Here, we reveal that these are not separated events, and the CYS-forming amyloid fibrils are mechanistically linked to hexagonal CTE crystals. For the first time, we demonstrated that cysteine fibrils are a prerequisite for forming cystine crystals, as observed experimentally. To further understand this mechanism, we studied the effects of thiol-containing cystinuria drugs (tiopronin, TIO; and d-penicillamine, PEN) and the canonical epigallocatechin gallate (EGCG) amyloid inhibitor on fibril formation by CYS. The thiol-containing drugs do not solely interact with monomeric CYS via disulfide bond formation but can disrupt amyloid formation by targeting CYS oligomers. On the other hand, EGCG forms inhibitor-dominant complexes (more than one EGCG molecule per cysteine unit) to prevent CYS fibril formation. Interestingly, while CYS can be oxidized into CTE, the thiol drugs can reduce CTE back to CYS. We thus suggest that the formation of crystals in cystinuria could be halted at the initial stage by targeting CYS fibril formation as an alternative to solubilizing the water-insoluble hexagonal CTE crystals at a later stage. Taken together, we depicted a complex hierarchical organization in a simple amino acid assembly with implications for therapeutic intervention.
Assuntos
Cisteína , Cistinúria , Humanos , Cisteína/química , Cistina/química , Cistinúria/tratamento farmacológico , Aminoácidos/uso terapêutico , Amiloide/química , Dissulfetos/uso terapêuticoRESUMO
While soluble forms of amyloid-ß (Aß) and Tau work together to drive healthy neurons into a disease state, how their interaction may control the prion-like propagation and neurotoxicity of Tau is not fully understood. The cross-linking via disulfide bond formation is crucial for Tau oligomers to obtain stable conformers and spread between cells. This work thus focuses on how Aß42 regulates this critical process. By studying the interactions between Aß42 and TauPHF43, a construct that mimics the Tau R3 isoform, has a similar length to Aß42, and contains one cysteine (Cys-322), we discovered that fresh Aß42 could protect Tau against the formation of disulfide cross-linked dimers. We showed that the monomeric and small Aß oligomers (the "nonamyloidogenic Aß") efficiently disassembled tau dimers and heparin-induced Tau oligomers to recover Tau monomers. Interestingly, Aß serves the role of an antioxidant to prevent disulfide bond formation, as supported by the experiments of Aß with cystine. Furthermore, using cyclosporine A (CycA), a macrocyclic ß-sheet disruptor, we demonstrated that targeting amyloidogenic Aß with CycA does not affect the TauPHF43 disassembly driven by Aß42. Separately, we assessed the initial toxicity of Aß42 and TauPHF43 in acute brain slices and found that Aß42 is more toxic than TauPHF43 or the two peptides combined. Our work highlights a potential protective role of Aß42 monomers in AD that was previously overlooked while focusing on the mechanism behind Aß42 aggregation leading to tau dysfunction.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Neurônios , Fragmentos de Peptídeos/químicaRESUMO
An atomic view of a main aqueous conformation of cyclosporine A (CycA), an important 11-amino-acid macrocyclic immunosuppressant, is reported. For decades, it has been a grand challenge to determine the conformation of free CycA in an aqueous-like solution given its poor water solubility. Using a combination of X-ray and single-crystal neutron diffraction, we unambiguously resolve a unique conformer (A1) with a novel cis-amide between residues 11 and 1 and two water ligands that stabilize hydrogen bond networks. NMR spectroscopy and titration experiments indicate that the novel conformer is as abundant as the closed conformer in 90/10 (v/v) methanol/water and is the main conformer at 10/90 methanol/water. Five other conformers were also detected in 90/10 methanol/water, one in slow exchange with A1, another one in slow exchange with the closed form and three minor ones, one of which contains two cis amides Abu2-Sar3 and MeBmt1-MeVal11. These conformers help better understand the wide spectrum of membrane permeability observed for CycA analogues and, to some extent, the binding of CycA to protein targets.
Assuntos
Ciclosporina , Metanol , Amidas/química , Ligação de Hidrogênio , Conformação Molecular , Conformação Proteica , Água/químicaRESUMO
Gas-phase ion-mobility spectrometry provides a unique platform to study the effect of mobile charge(s) or charge location on collisional cross section and ion separation. Here, we evaluate the effects of cation/anion adduction in a series of xylene and pyridyl macrocycles that contain ureas and thioureas. We explore how zinc binding led to unexpected deprotonation of the thiourea macrocyclic host in positive polarity ionization and subsequently how charge isomerism due to cation (zinc metal) and anion (chloride counterion) adduction or proton competition among acceptors can affect the measured collisional cross sections in helium and nitrogen buffer gases. Our approach uses synthetic chemistry to design macrocycle targets and a combination of ion-mobility spectrometry mass spectrometry experiments and quantum mechanics calculations to characterize their structural properties. We demonstrate that charge isomerism significantly improves ion-mobility resolution and allows for determination of the metal binding mechanism in metal-inclusion macrocyclic complexes. Additionally, charge isomers can be populated in molecules where individual protons are shared between acceptors. In these cases, interactions via drift gas collisions magnify the conformational differences. Finally, for the macrocyclic systems we report here, charge isomers are observed in both helium and nitrogen drift gases with similar resolution. The separation factor does not simply increase with increasing drift gas polarizability. Our study sheds light on important properties of charge isomerism and offers strategies to take advantage of this phenomenon in analytical separations.
Assuntos
Hélio , Xilenos , Ânions , Isomerismo , Metais/química , Nitrogênio/química , Prótons , ZincoRESUMO
High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated ß-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between ß-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Homocisteína/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Homocisteína/química , Humanos , Espectrometria de Mobilidade Iônica , Cinética , Camundongos Transgênicos , Modelos Biológicos , Polifenóis/farmacologia , Saccharomyces cerevisiae/metabolismoRESUMO
Our knowledge of amyloid formation and cytotoxicity originating from self-assembly of α-helical peptides is incomplete. PSMα3 is the only system where high-resolution X-ray crystallography and toxicity data are available. Oligomers of multiple α-helical monomers are less stable than those of ß-strands, partially due to the lack of a consistent hydrogen-bonding network. It is challenging to preserve such oligomers in the gas phase where mass-selected structural studies using ion-mobility spectrometry mass spectrometry (IMS-MS) could be performed. As the oligomers fall apart after exiting the drift cell of the mass spectrometer, novel features that have shorter (a loss of charged species) or longer (a loss of neutral species) arrival times than expected are present together with those from the intact species. By obtaining a complete data set of PSMα3 peptides in solution and with n-dodecyl-ß-d-maltoside, a micelle-forming detergent, we are able to discern the dissociated from the intact oligomers and detergent-bound complexes and correlate the reported cytotoxicity to the peptide oligomeric structures and their interactions with membrane mimetics. The study sheds new insights into the interpretation of IMS-MS data from biomolecular self-assembly studies-an important and timely topic.