Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

This work was supported by Instituto de Salud Carlos III (FIS-PI10-00975), Generalitat de Catalunya (SGR 2009-00761), FEDER (BIO2011-22568) and MICIN (BES-2009-024653). G.R-H is supported by a MAEC-AECI fellowship and M.M-A by a PIF (UAB) fellowship.Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either eningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control

Estudi de la via d’agregació de tres proteïnes implicades en diferents malalties conformacionals humanes: scFv‐h3D6 com agent terapèutic per a la malaltia d’Alzheimer, AL‐12 com a causa d’Amiloïdosi de cadena lleugera, i PDZ3 com a organitzador del proteoma

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

Estudi de la via d’agregació de tres proteïnes implicades en diferents malalties conformacionals humanes: scFv‐h3D6 com agent terapèutic per a la malaltia d’Alzheimer, AL‐12 com a causa d’Amiloïdosi de cadena lleugera, i PDZ3 com a organitzador del proteoma

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

Estudi de la via d'agregació de tres proteïnes implicades en diferents malalties conformacionals humanes : scFv-h3D6 com agent terapèutic per a la malaltia d'Alzheimer, AL-12 com a causa d'amiloïdosi de cadena lleugera, i PDZ3 com a organitzador del proteoma

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

The Impact of Extra-Domain Structures and PostTranslational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2-β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2-β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

This work was supported by Instituto de Salud Carlos III (FIS-PI10-00975), Generalitat de Catalunya (SGR 2009-00761), FEDER (BIO2011-22568) and MICIN (BES-2009-024653). G.R-H is supported by a MAEC-AECI fellowship and M.M-A by a PIF (UAB) fellowship.Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either eningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest