Search of ligands for the amyloidogenic protein beta2-microglobulin by capillary electrophoresis and other techniques

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.     

33 Hz脉冲电场辐照对胰岛素淀粉样变性的影响

蛋白质结构失稳后会发生错误折叠、集聚或纤维化,不仅丧失正常功能,有时甚至会产生生物毒性,导致相关疾病。纤维化了的蛋白质是神经退行性疾病以及二型糖尿病等的主要诱因,它在溶液中具有和淀粉类似的特性,因此蛋白质的纤维化又被称为淀粉样变性。电磁辐射是引起蛋白质结构失稳的一个常见因素。经电场辐照后,蛋白质会发生去折叠和集聚,改变自发折叠机制。同时,经受电场辐照的扰动而失稳的蛋白质,纤维化过程也会受到一定的影响。以胰岛素为研究对象,利用硫磺素T(ThT)染色的荧光光谱法、透射电子显微镜(TEM)以及圆二色谱(CD)法,分别从纤维化的宏观动力学过程、成熟纤维丝微观形貌以及原纤维二级结构组成等不同角度,探究经33 Hz脉冲电场(PEF)不同电场强度及不同持续时间辐照后蛋白质在体外淀粉样变性机制的变化情况。结果表明,胰岛素溶液经过33 Hz PEF辐照后,淀粉样变性初期产生前体蛋白的成核期延长,原纤维丝聚集延长过程(即中间产物的寿命)缩短,形成的成熟纤维丝长度变短且整齐成簇无分支。这些效应随着辐照电场强度和辐照时间的增加而有不同程度的加强。研究结果一致说明PEF辐照对胰岛素的淀粉样变性有一定的抑制作用。     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Evaluation of capillary electrophoresis-mass spectrometry for the analysis of the conformational heterogeneity of intact proteins using beta2-microglobulin as model compound

In this work we explored the feasibility of different CE-ESI-MS set-ups for the analysis of conformational states of an intact protein. By using the same background electrolyte at quasi physiological conditions (50 mM ammonium bicarbonate, pH 7.4) a sequential optimization was carried out, initially by evaluating a sheath-liquid interface with both a single quadrupole (SQ) and a time-of-flight (TOF) mass spectrometer; then a sheathless interface coupled with high-resolution QTOF MS was considered. Beta₂-microglobulin has been taken as a model, as it is an amyloidogenic protein and its conformational changes are strictly connected to the onset of a disease. The separation of two conformers at dynamic equilibrium is achieved all the way down to the MS detection. Notably, the equilibrium ratio of the protein conformers is maintained in the electrospray source after CE separation. Strengths and weaknesses of each optimized set-up are emphasized and their feasibility in unfolding studies is evaluated. In particular, ESI-TOF MS can assign protein forms that differ by 1 Da only and sheathless interfacing is best suited to preserve protein structure integrity. This demonstrates the CE-ESI-MS performance in terms of separation, detection and characterization of conformational species that co-populate a protein solution.     

Oxidatively-mediated in silico epimerization of a highly amyloidogenic segment in the human calcitonin hormone (hCT15-19)

In order to study the effects of peptide exposure to oxidative attack, we chose a model reaction in which the hydroxyl radical discretely abstracts a hydrogen atom from the α-carbon of each residue of a highly amyloidogenic region in the human calcitonin hormone, hCT_15-19. Based on a combined Molecular Mechanics / Quantum Mechanics approach, the extended and folded L- and D-configuration and radical intermediate hCT_15-19 peptides were optimized to obtain their compactness, secondary structure and relative thermodynamic data. The results suggest that the epimerization of residues is generally an exergonic process that can explain the cumulative nature of molecular aging. Moreover, the configurational inversion induced conformational changes can cause protein dysfunction. The epimerization of the central residue to the D-configuration induced a hairpin structure in hCT_15-19, concomitant with a possible oligomerization of human calcitonin into Aβ(1–42)-like amyloid fibrils present in patients suffering from Alzheimer’s disease.     

Immunoaffinity chromatographic and immunoprecipitation methods combined with mass spectrometry for characterization of circulating transthyretin

Transthyretin (TTR) is a small serum protein that is involved in distinct phenotypes of amyloidosis with different tissue localization, age of onset, and rate of progression. Some types of TTR-amyloidosis (such as familial amyloid polyneuropathy) are associated with various amino acid point mutations, while cardiac amyloid myopathy may also be associated with precipitation of the wild-type molecules, e.g., in senile systemic amyloidosis. Because of the unsettled relationship between circulating and precipitated TTR we here explore on-line immunoaffinity (IA) chromatography-MS and immunoprecipitation (IP)-MS methods for characterizing the circulating TTR population in normal individuals and in patients with known TTR-amyloidosis. It was found necessary to reduce the samples, e.g., with DTT, prior to ESI-TOF-MS. This reversed oxidative modifications to sufficiently resolve the two mass peaks isolated from sera of heterozygous patients. A simple IP technique without the use of centrifugal filtration was found to be convenient for the assessment of the TTR population in serum as demonstrated for both normal and variant (the Met111Leu mutation) TTR. This approach also readily allowed the identification of oxidation, S-sulfation, and S-cysteinylation in unreduced samples, while these modifications were less well resolved in the on-line IA chromatography-MS approach.     

Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2‐microglobulin as a simple tool for misfolding and stability studies

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild‐type β₂‐microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25–42°C is induced inside the capillary for the two pathogenic proteins (wtβ₂‐microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.