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Aquesta Tesi Doctoral es centra en l’estudi molecular de tres proteïnes relacionades amb malalties conformacionals humanes, o Amiloïdosis. Mitjançant tècniques espectroscòpiques, s’han descrit els mecanismes de plegament natiu i d’agregació en fulla-β ordenada, dues vies que es troben en competència a través de la presència de diferents estats intermediaris a l’espai conformacional. Els intermediaris d’agregació, precursors de les fibres amiloides, són considerats els principals causants de la citotoxicitat cel·lular en moltes de les malalties amiloidogèniques, per tant, el seu estudi és crucial per aprofundir en l’etiologia d’aquestes patologies humanes, moltes d’elles sense tractament curatiu. Per una banda, s’ha obtingut un fragment d’anticòs recombinant específic per a β-oligòmers del pèptid Aβ1-42 causant de l’Alzheimer, l’scFv-h3D6. El seu estudi ha estat el primer en descriure el mecanisme pel qual un scFv retira el pèptid Aβ1-42 de la via amiloide i evita la seva toxicitat neuronal, tal i com s’ha demostrat en assajos de viabilitat en cultius cel·lulars. A més, l’obtenció d’un model tridimensional de l’scFv-h3D6, per homologia de seqüències, ha permès fer un redisseny racional de la molècula per tal de millorar-ne la seva estabilitat, augmentar la seva producció, i disminuir la dosi efectiva ja demostrada en ratolins model d’Alzheimer. Per altra banda, s’ha treballat amb un domini variable (VL) d’immunoglobulina, extret d’una pacient amb Amiloïdosi de cadena lleugera, l’AL-12. Aquest domini amiloidogènic presenta 7 mutacions respecte la seva línia germinal κI O18/O8. L’obtenció de les propietats termodinàmiques de l’AL-12, així com de quatre mutants restauratius, ha servit per estudiar l’efecte de les mutacions somàtiques sobre l’estabilitat dels dominis VL. Així, la restauració de residus puntuals de l’AL-12 cap a la seqüència germinal, implica la desestabilització del domini i un augment de la tendència a l’agregació i, per tant, s’ha demostrat que una única mutació somàtica no seria la causa del potencial amiloidogènic de l’AL-12, sinó que seria la combinació de varis factors desestabilitzants del plegament natiu, donant lloc a canvis conformacionals susceptibles a l’agregació amiloide, i la seva deposició en diferents òrgans i teixits cel·lulars. I per últim, s’ha estudiat el domini PSD95-PDZ3, una molècula reguladora de la sinapsis neuronal, i relacionada amb malalties com l’Alzheimer o l’Autisme. El domini PDZ3 segueix una via d’agregació amiloide reversible, una particularitat rellevant de la qual se n’han determinat els canvis conformacionals que la promouen. A més, el disseny de 6 variants del PDZ3 han servit per determinar les regions implicades en l’estabilitat i plegament global del domini, així com el seu paper en la regulació al·lostèrica de l’activitat del domini. Aquest treball demostra la importància dels estats intermediaris a l’espai conformacional de les proteïnes, la seva relació amb les malalties conformacionals, i l’aplicabilitat del seu coneixement en el desenvolupament de teràpies efectives., The thesis here presented is focused on the molecular study of three human proteins related to conformational diseases, or Amyloidoses. The mechanisms of native folding and aggregation in a cross-β sheet structure have been described using several spectroscopic techniques. These two pathways are competing by the presence of intermediate states of the reaction. An oligomeric intermediate is the precursor of the β-aggregates and is considered the main responsible of the cellular toxicity in many amyloid diseases. Therefore, is necessary to perform a deeper study of the oligomeric state to delve into the etiology of these human diseases. On one hand, the expression and extraction of a single-chain variable fragment (scFv-h3D6), a derivative of an antibody specific for Aβ-oligomers, was performed. Here is the first description of a conformational mechanism by which a scFv-h3D6 withdraws Aβ1–42 oligomers from the amyloid pathway and, consistently, it prevents Aβ-oligomer toxicity in the SH-SY5Y human neuroblastoma cell line. Moreover, we modeled the 3D-structure of scFv-h3D6, obtained by sequence homology. This model has allowed to a rational redesign of the molecule in order to improve its stability, increase its recombinant production, and decrease the effective dose already demonstrated in a mice model of Alzheimer's disease. For another hand, we studied the AL-12 protein, an Ig variable domain (VL) obtained from a patient diagnosed with light chain Amyloidoses. This amyloidogenic domain shows seven non-conservative mutations from his germinal protein κI O18/O8. The thermodynamic study of AL-12 and four restoratives mutants, allowed us to determine the effect of somatic mutations on the VL stability. Thereby, the restoration of a residue toward the germinal sequence implies a destabilization of the domain and increases the aggregation tendency. Here we demonstrate that a single somatic mutation couldn’t be the cause of the amyloidogenic potential of AL-12, rather it is due to several factors. The destabilization of the native folding promotes a conformational reorganization more susceptible to β-aggregation. Finally, we work with PSD95-PDZ3 domain, a neuronal post-synaptic protein and related to human disease such as Alzheimer or Autism. PDZ3 follows a reversible aggregation pathway, a feature which is relevant to determining the conformational changes that promote it. In addition, the design of six variants of PDZ3 has helped determine the regions involved in the folding and stability of the global PDZ3 domain and its role in the aggregation process. This work demonstrates the importance of the states in the intermediate conformational space of proteins, their relationship with conformational diseases, and the application of knowledge in the development of effective therapies.

This work was supported by FMM-2008; FEDER (FISPI10-00975, -00265 and -00283); SGR2009-00761 and -42271. G.R-H is supported by a MAEC-AECI fellowship (Spanish government) and M.M-A by a PIF (UAB, Spain) fellowship. The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid β (Aβ) peptide in neuroblastoma cell cultures by withdrawing Aβ oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia -like behaviors. Cognitive deficits included long- and short-term learning and memory deficits and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aβ oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.

Background: Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell clonal proliferation. Currently, the best biomarker for treatment monitoring is serum free light chain (FLC) assay. Despite its higher sensitivity, it cannot distinguish clonality thus ultralow levels of monoclonal gammopathy can be missed. Urinary exosomes (UExs) are the smallest members of the extracellular vesicle family that are excreted in the urine. They exhibit different characteristics in patients with AL amyloidosis vs multiple myeloma. This study was undertaken to determine if urinary exosomes have different characteristics in patients AL amyloidosis who progress despite being in complete response (CR) vs those who have a renal response. Methods: UEx were extracted from 4 patients at different stages of AL (Table 1). Immunoglobin light chains (LC) were identified in the UEx using western blotting. Intact immunoglobulin light chains were identified in plasma, UEx, and kidney biopsy amyloid deposits using mass spectrometry (MS). cDNAs from bone marrow plasma cells (BMPC) collected at the time of diagnosis from patient 4 were sequened. Results: Oligomeric LC (250 kd) were identified in the UEx of patient 1 (newly diagnosed, Fig 1a) and 4 (renal progression in CR, Fig 1d) but only monomeric LC (25 kd) were detected in patient 2 (Fig 1b) and 3 (CR and renal response, Fig 1c). MS of the UEx and plasma of patient 4 detected 2 monoclonal λ LC. The mass of one of the LC was consistent with a lambda 6a LC which is also the most common cDNA (IGLV-6-57) found in the BMPC. Amino acid sequences derived from the tryptic digestion of the amyloid deposits matched the predicted sequence of the cDNA. The calculated weight of the peptide produced by the cDNA (23,304 Da ) was within margin of error of the mass of the lambda 6a LC (23,306 Da). The mass of the second LC (23,092 Da) was consistent with a λ4/λ5 LCs. A search of the amino acid and cDNA sequences failed to identify any similarity. The density ratio of the oligomeric LC to monomeric LC in the UEx was 0.47 which was similar to the intensity ratio by MS of the lambda 6a LC (23,306 Da) to the 23,092 Da LC in the blood (0.43). Discussion: Urinary EXs identified oligomeric LC in a patient with progressive renal disease despite achieving a CR. The mass one of the λ LC matched the predicted peptide product of the cDNA obtained from BMPC. The amino acid sequence predicted by the cDNA matched the trypsin digested sequences from the amyloid deposits. This strongly suggests the oligomeric LC represents the amyloidogenic λ6a LC detected in the blood which was being produced by the BMPC clone which is being deposited as amyloid in the kidney. The origin of the second λ LC is unknown but is likely a new monoclonal gammopathy of undetermined significance that developed after the initial bone marrow biopsy. The blood and urine samples were obtained 3 years after the BMPC. The development of a new monoclonal gammopathy in patients with plasma cell dyscrasia is not uncommon. Unfortunately, bone marrow biopsy collected at the time of the urine and blood sample had insufficient number of plasma cells (CR) for analysis. Conclusion: UEx can identify amyloidogenic LC even when current standard methods could not. The persistent presence of the amyloidogenic LC helps explain why patients can progress despite being in CR. The presence of a second λ LC which was not part of the amyloid deposit also has clinical implication. It may explain why some patients do well despite having a persistent monoclonal protein. If these results are confirmed, UEx may have a powerful role in the determination of organ and hematologic response in patients with AL amyloidosis. Disclosures Dispenzieri: Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; pfizer: Research Funding; Jannsen: Research Funding; Celgene: Research Funding.