Ersilia De Lorenzi

Selectivity in capillary electrophoresis: the use of proteins

Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.

Surface initiated molecularly imprinted polymer films: a new approach in chiral capillary electrochromatography

A new generation of imprinted composite particles was tested as capillary electrochromatography stationary phase. Silica particles characterised by a well defined particle size (10 µm diameter), shape and pore system (1000 A) were modified with an azoinitiator and subsequently used to graft molecularly imprinted polymers targeted to bind L-phenylalanine anilide. Fused silica capillaries were packed over a length corresponding to 8 cm, using a pneumatic amplification pump, and the stationary phase thus obtained was tested with respect to its electrochromatographic performance. The electroendosmotic flow mobility was evaluated with respect to both the different content of polymer on the silica particle surface and different operating pH values. The dependence of various parameters, namely the analyte concentration, the polymer layer thickness and the pH of the mobile phase on the enantioselectivity was investigated. These CEC capillaries showed enantioselectivity comparable with that showed in LC, and exhibited improved performance in terms of plate number N1 (∼13 000), selectivity α (∼1.5), analysis time (<3 min), inter–intraday and intercapillary reproducibility. We expect this approach to result in a new generation of robust, tailor-made chiral or affinity stationary phases for CEC.

A Phosphotyrosine‐Imprinted Polymer Receptor for the Recognition of Tyrosine Phosphorylated Peptides

Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Dialysis‐related amyloidosis is a disease in which partial unfolding of β2‐microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of β2‐microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full‐length β2‐microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full‐length β2‐microglobulin.

Pharmaceutical strategies against amyloidosis: Old and new drugs in targeting a protein misfolding disease

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, proteins post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.

CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity

Soluble and toxic oligomers of amyloid β (Aβ) protein have been identified as the true neurotoxic species involved in Alzheimers disease and considering them as targets to inhibit Aβ aggregation might have a therapeutic value. We previously set up a CE method that enables the separation and quantification of transient oligomers of Aβ protein‐containing 42 amino acids (Aβ1–42) along the pathway leading to fibrils and we now demonstrate how this method can be successfully applied to examine the in vitro inhibitory effects of small molecules on Aβ oligomerization. To this end, we investigated mitoxantrone and pixantrone, two well‐known anticancer drugs, as well as suramin and a suramin‐like compound. By using CE, it is here shown how mitoxantrone and pixantrone either reduce or block Aβ1–42 oligomerization, while Thioflavin T spectrofluorimetric assay and transmission electron microscopy demonstrate how these two compounds also display antifibrillogenic activity. Interestingly, in vitro cell viability experiments indicated that pixantrone significantly reduces Aβ1–42 neurotoxicity.

Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation.

In order to identify novel Alzheimers modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Aβ). Accordingly, compounds 2a-c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Aβ aggregation and with the Aβ self-oligomerization process (2a). Compounds 2a-c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Aβ aggregation since they physically hamper Aβ binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Aβ self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Aβ aggregation.

Disease modifying anti-Alzheimer’s drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation

We recently described multifunctional tools (2a–c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level.

In vitro amyloid Aβ1-42 peptide aggregation monitoring by asymmetrical flow field-flow fractionation with multi-angle light scattering detection

Self-assembly of the 42-amino-acid-long amyloid peptide Aβ1-42 into insoluble fibrillar deposits in the brain is a crucial event in the pathogenesis of Alzheimers disease. The fibril deposition occurs through an aggregation process during which transient and metastable oligomeric intermediates are intrinsically difficult to be accurately monitored and characterised. In this work, the time-dependent Aβ1-42 aggregation pattern is studied by asymmetrical flow field-flow fractionation with on-line multi-angle light scattering detection. This technique allows separating and obtaining information on the molar mass (Mr) and size distribution of both the early-forming soluble aggregates and the late prefibrillar and fibrillar species, the latter having very high Mr. Preliminary results demonstrate that unique information on the dynamic aggregation process can be obtained, namely on the Mr and size of the forming aggregates as well as on their formation kinetics.

Variants of β2-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

Cleavage of the small amyloidogenic protein β2‐microglobulin after lysine‐58 renders it more prone to unfolding and aggregation. This is important for dialysis‐related β2‐microglobulin amyloidosis, since elevated levels of cleaved β2‐microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved β2‐microglobulin variants have not yet been assessed using single‐residue techniques. We here use such methods to examine β2‐microglobulin cleaved after lysine‐58 and the further processed variant (found in vivo) from which lysine‐58 is removed. We find that the solution stability of both variants, especially of β2‐microglobulin from which lysine‐58 is removed, is much reduced compared to wild‐type β2‐microglobulin and is strongly dependent on temperature and protein concentration. 1H‐NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three‐dimensional structure of the variants is similar to that of wild‐type β2‐microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E β‐strands close to the D–E loop cleavage site at lysine‐58, and the experiments suggest conformational heterogeneity of the two variants. Two‐dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native‐like fold and at least one conformational intermediate resembling intermediates found in other structurally altered β2‐microglobulin molecules. This is the first single‐residue resolution study of a specific β2‐microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in β2‐microglobulin amyloidogenicity in vivo.