A biophysical approach to study an orphan disease: the case of CblC, a rare disorder of vitamin B12 intracellular metabolism

The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism. The affected children manifest devastating symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a protein that transports and transforms the different Cbl forms. Although the crystal structure of the wild-type (WT) protein is available, a systematic study on the effect of each specific mutation on the resulting protein is still lacking. Here we present data on the biophysical characterization of WT MMACHC, and two variants resulting from CblC pathological mutations. By using a biophysical approach including spectroscopy, Dynamic-Static Light and Small X-Ray Angle Scattering, and Molecular Dynamics, we investigated protein structure/stability and ability to bind and transform Cbl. Moreover, we evaluated whether drug-like molecules identified by computational methods, or non-specific stabilizers (osmolytes) could restore functionality in MMACHC mutants. Overall our results reveal how a biophysical approach can offer new insights in the study of CblC mutations' specific effect and help prospecting new routes for the CblC treatment

The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway

The Tempered Polymerization of Human Neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation

Simple model systems: a challenge for Alzheimer's disease

The success of biomedical researches has led to improvement in human health and increased life expectancy. An unexpected consequence has been an increase of age-related diseases and, in particular, neurodegenerative diseases. These disorders are generally late onset and exhibit complex pathologies including memory loss, cognitive defects, movement disorders and death. Here, it is described as the use of simple animal models such as worms, fishes, flies, Ascidians and sea urchins, have facilitated the understanding of several biochemical mechanisms underlying Alzheimer's disease (AD), one of the most diffuse neurodegenerative pathologies. The discovery of specific genes and proteins associated with AD, and the development of new technologies for the production of transgenic animals, has helped researchers to overcome the lack of natural models. Moreover, simple model systems of AD have been utilized to obtain key information for evaluating potential therapeutic interventions and for testing efficacy of putative neuroprotective compounds

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer’s disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting

Lower bounds and heuristics for the Windy Rural Postman Problem

[EN] In this paper we present several heuristic algorithms and a cutting-plane algorithm for the Windy Rural Postman Problem. This problem contains several important Arc Routing Problems as special cases and has very interesting real-life applications. Extensive computational experiments over different sets of instances are also presented.The contribution by E. Benavent, A. Corberán and J.M. Sanchis has been supported by the Ministerio de Ciencia y Tecnología of Spain through grant TIC2003-05982-C05-01 (partially financed with FEDER funds) and by the Generalitat Valenciana (Ref: GRUPOS03/174 and GRUPOS03/ 189). A. Carrotta and D. Vigo thank the Ministero Dell Istruzione, dell Università e della Ricerca (M.I.U.R.) and the Consiglio Nazionale delle Ricerche (C.N.R.), Italy, for the support given to this project.Benavent, E.; Carrotta, A.; Corberán, A.; Sanchís Llopis, JM.; Vigo, D. (2020). Lower bounds and heuristics for the Windy Rural Postman Problem. European Journal of Operational Research. 176(2):855-869. https://doi.org/10.1016/j.ejor.2005.09.021S855869176