Big dynorphin is a neuroprotector scaffold against amyloid β-peptide aggregation and cell toxicity

Amyloid β-peptide (Aβ) misfolding into β-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aβ aggregation are highly relevant as possible AD treatments since they should protect against Aβ neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aβ40 the most abundant species of Aβ. Biophysical measurements indicate that Aβ40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aβ40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aβ40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.The authors would like to thank Mr. Jordi Pujols Pujol for skillful technical assistance in RP-HPLC experiments, and Mr. Mateo Calle Velásquez for skillful assistance in the docking process.Peer reviewe

Human Albumin Impairs Amyloid β-peptide Fibrillation Through its C-terminus: From docking Modeling to Protection Against Neurotoxicity in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative process characterized by the accumulation of extracellular deposits of amyloid β-peptide (Aβ), which induces neuronal death. Monomeric Aβ is not toxic but tends to aggregate into β-sheets that are neurotoxic. Therefore to prevent or delay AD onset and progression one of the main therapeutic approaches would be to impair Aβ assembly into oligomers and fibrils and to promote disaggregation of the preformed aggregate. Albumin is the most abundant protein in the cerebrospinal fluid and it was reported to bind Aβ impeding its aggregation. In a previous work we identified a 35-residue sequence of clusterin, a well-known protein that binds Aβ, that is highly similar to the C-terminus (CTerm) of albumin. In this work, the docking experiments show that the average binding free energy of the CTerm-Aβ1–42 simulations was significantly lower than that of the clusterin-Aβ1–42 binding, highlighting the possibility that the CTerm retains albumin's binding properties. To validate this observation, we performed in vitro structural analysis of soluble and aggregated 1 μM Aβ1–42 incubated with 5 μM CTerm, equimolar to the albumin concentration in the CSF. Reversed-phase chromatography and electron microscopy analysis demonstrated a reduction of Aβ1–42 aggregates when the CTerm was present. Furthermore, we treated a human neuroblastoma cell line with soluble and aggregated Aβ1–42 incubated with CTerm obtaining a significant protection against Aβ-induced neurotoxicity. These in silico and in vitro data suggest that the albumin CTerm is able to impair Aβ aggregation and to promote disassemble of Aβ aggregates protecting neurons

Expression and characterization of a human sodium glucose transporter (hSGLT1) in Pichia pastoris

En els últims 20 anys, la caracterització de proteines de membrana ha esdevingut un camp interessant per el disseny de fàrmacs però, la falta d’informació del seu mecanisme d’acció i estructura fa encara més difícil buscar noves dianes terapèutiques. Per tant, estudiar l’estructura i el mecanisme d’acció de proteines de membrana acceleraria la recerca de nous fàrmacs i les seves implicacions mèdiques. El projecte de tesis presentat aquí està centrat en explorar les característiques del transprotador de glucosa humà (hSGLT1). Aquest transportador està implicat en la absorció de glucosa intestinal i està relacionat directament amb diferents enfermetats com: glucosa galactose malabsorbation (GGM), diabetis i l’enfermetat d’Alzheimer. El mecanisme de funcionament d’aquest transportador esta parcialment caracteritzat malgrat que algunes característiques encara no estan clares i s’estan estudiant. No obstant, l’estructura tridemensional del transport no esta descrita i, actualment, és el màxim objectiu pel que fa a entendre com funciona el seu mecanisme i que permetria buscar més fàcilment i efectivament nous fàrmacs. Inicialment, per tal d’obtenir un cristall de proteïna per poder resoldre la seva estructura, es necessiten un gran número de pasos previs. Primer, la proteina d’interest ha de ser expressada en un sistema recombinant correctament i, les proteïnes de membrana no són fàcils d’expressar en tots els sistemes d’expressió. En segon lloc, les proteïnes de membrana humanes son encara més complicades d’expressar perquè es necessari treballar en un sistema d’expressió eucariote o, sinó, es molt probable que s’obtingui una proteïna funcionalment inactiva. hSGLT1 ha sigut obtingut recombinant només un cop per un sistema eucariote de llevat: Pichia pastoris. Aquest llevat ha sigut utilitzat en els últims 10 anys per expressar proteines de membrana perque, a diferència d’altres llevats, com Saccharomyces cerevisae, pot crèixer molt més i, per tant, obtenir molta més proteïna. Una altre avantatge d’aquest sistema d’expressió és l’estabilitat dels clons de P. pastoris ja que implica més reproducibilitat en l’expressió. Tot i així, algunes desavantatges presenta aquest sistem que s’han de superar per tal d’obtenir bons nivells d’epressió, com per exemple, la selecció de clons. Degut a tot aixó, el projecte que es presenta aquí esta basat inicialment amb els treballs previs realitzats amb P. pastoris. Aquesta tesis esta centrada en l’expressió, purificació i caracterització del transportador de glucosa humà (hSGLT1) expressat en un sistema d’expressió heteroluga de llevat (P.pastoris). Inicialment, dos vectors es van dissenyar: Un que expressa hSGLT1 fusionat amb eGFP i l’altre que no (WT). El constructe que expresa la proteina fusionada amb eGFP es va utilitzar per monotiroritzar l’expressió de manera més fàcil i ràpida i que, per tant, permet optimitzar les condicions d’expressió, com per exemple: temps d’inducció, temperature, medis.etc. Les proteines de membrana necessiten ser extrete de la membrana per poder-les purificiar i, per tant, es requereix de detergent. El constructe amb eGFP es va utilitzar també per buscar quin detergent era més òptim per extreure la proteïna. Tot i així, tots ambdós costructes (WT i eGFP) es van purificar i aïllar correctament però només el producte WT es va utilitzar per els següents experiments estructurals. Malgrat les dificultats de reproduir el que s’havia descrit anteriorment, la proteina finalment es va aconseguir sotmetre a assaijos de funcionalitat. L’assaig de binding es va fer a partir d’una tècnica inusual: voltage-clamp in planar lipid membranes. Aquesta tècnica permet mesurar transport (funció) del transportador sense la necessitat d’incorporarar-la en liposomes. La conclusió final que es pot extreure en aquest treball es que hSGLT1 es pot purificar correctament i que, per tant, obre un gran ventall de possibilitats en un futur, com per exemple, cristalitzar la proteina per tal de resoldre la seva estructura i el seu mecanisme.In the last 20 years, the characterization of membrane protein has become an interesting field for drug design but, the lack of information regarding their mechanism of action makes it even harder to screen for new drug targets. Therefore, studying the structure and mechanism of action of membrane proteins should accelerate the research of new drugs and its medical implications. The project presented here is focused on exploring the features of a human sodium glucose co-transporter (hSGLT1). This transport is involved in absorbing and reabsorbing the glucose in the intestine and it’s linked and directly involved in some diseases like: glucose galactose malabsorbation (GGM), diabetes and even some mental diseases like Alzheimer’s diseases. The mechanism of action of this transporter is partially understood although some features are still under debate and unclear. However, the 3D structure of this transporter is unknown and it’s an end goal for elucidating the mechanism of action of the transporter which will allow screening easily more effective drugs. In order to be available to crystalize a membrane protein for its structure, a huge number of initial steps are necessary. First, the protein of interest must be expressed in a recombinant system satisfactorily and, membrane proteins are not easily expressed in all expression systems. Second, human membrane proteins are even harder to express because it’s necessary to work with a non-prokaryote expression system or, otherwise, it is very likely to obtaining in the end a non-functional protein. It has been described, only once, that hSGLT1 could be expressed in a eukaryotic yeast expression system: Pichia pastoris. This yeast has been used a lot in the last 10 years for expressing membrane proteins successfully because, unlike Saccharomyces cerevisae, it can grow much more and therefore obtain more protein in the end. Another advantage is the stability of the expression clones of P. pastoris which eventually leads to a more reproducible result. Although, some disadvantages must be overcome at the same time to have good expression levels like, for example, the clone selection. Because of all of this, the project presented here was initially based on this previous work done in P. pastoris. This thesis is focused on the expression, purification and characterization of hSGLT1 expressed in a yeast heterologous expression system (P.pastoris). Initially, two vectors were design: One expressing hSGLT1 fused to eGFP and another construct without eGFP. The eGFP construct was used to monitorize the protein expression faster and easier which allows optimizing the protein expression by screening for best expression conditions like: induction time, temperature, media and more. Membrane proteins required to be extracted from the membrane in order to purify them and, to do so; a detergent is required. The eGFP fused protein was also used to screen which detergent is more suitable for protein extraction too. Although, both expression product (non-GFP and GFP) were purified and isolated only the WT hSGLT1 was used for further analysis. Despite the difficulties to reproduce what was described before, the protein was eventually subjected to a binding assay. The binding assay was done with an unusual technique which is: voltage-clamp in planar lipid membranes. This technique allowed to measure transport (functionality) of the protein without the need to be incorporated in a liposomes. The main conclusion extracted in this work is that a functional hSGLT1 can be purified which allows a wide number of possibilities in the future like crystallization for elucidating the structure and mechanism of the protein

Expression and characterization of a human sodium glucose transporter (hSGLT1) in Pichia pastoris /

En els últims 20 anys, la caracterització de proteines de membrana ha esdevingut un camp interessant per el disseny de fàrmacs però, la falta d'informació del seu mecanisme d'acció i estructura fa encara més difícil buscar noves dianes terapèutiques. Per tant, estudiar l'estructura i el mecanisme d'acció de proteines de membrana acceleraria la recerca de nous fàrmacs i les seves implicacions mèdiques. El projecte de tesis presentat aquí està centrat en explorar les característiques del transprotador de glucosa humà (hSGLT1). Aquest transportador està implicat en la absorció de glucosa intestinal i està relacionat directament amb diferents enfermetats com: glucosa galactose malabsorbation (GGM), diabetis i l'enfermetat d'Alzheimer. El mecanisme de funcionament d'aquest transportador esta parcialment caracteritzat malgrat que algunes característiques encara no estan clares i s'estan estudiant. No obstant, l'estructura tridemensional del transport no esta descrita i, actualment, és el màxim objectiu pel que fa a entendre com funciona el seu mecanisme i que permetria buscar més fàcilment i efectivament nous fàrmacs. Inicialment, per tal d'obtenir un cristall de proteïna per poder resoldre la seva estructura, es necessiten un gran número de pasos previs. Primer, la proteina d'interest ha de ser expressada en un sistema recombinant correctament i, les proteïnes de membrana no són fàcils d'expressar en tots els sistemes d'expressió. En segon lloc, les proteïnes de membrana humanes son encara més complicades d'expressar perquè es necessari treballar en un sistema d'expressió eucariote o, sinó, es molt probable que s'obtingui una proteïna funcionalment inactiva. hSGLT1 ha sigut obtingut recombinant només un cop per un sistema eucariote de llevat: Pichia pastoris. Aquest llevat ha sigut utilitzat en els últims 10 anys per expressar proteines de membrana perque, a diferència d'altres llevats, com Saccharomyces cerevisae, pot crèixer molt més i, per tant, obtenir molta més proteïna. Una altre avantatge d'aquest sistema d'expressió és l'estabilitat dels clons de P. pastoris ja que implica més reproducibilitat en l'expressió. Tot i així, algunes desavantatges presenta aquest sistem que s'han de superar per tal d'obtenir bons nivells d'epressió, com per exemple, la selecció de clons. Degut a tot aixó, el projecte que es presenta aquí esta basat inicialment amb els treballs previs realitzats amb P. pastoris. Aquesta tesis esta centrada en l'expressió, purificació i caracterització del transportador de glucosa humà (hSGLT1) expressat en un sistema d'expressió heteroluga de llevat (P.pastoris). Inicialment, dos vectors es van dissenyar: Un que expressa hSGLT1 fusionat amb eGFP i l'altre que no (WT). El constructe que expresa la proteina fusionada amb eGFP es va utilitzar per monotiroritzar l'expressió de manera més fàcil i ràpida i que, per tant, permet optimitzar les condicions d'expressió, com per exemple: temps d'inducció, temperature, medis.etc. Les proteines de membrana necessiten ser extrete de la membrana per poder-les purificiar i, per tant, es requereix de detergent. El constructe amb eGFP es va utilitzar també per buscar quin detergent era més òptim per extreure la proteïna. Tot i així, tots ambdós costructes (WT i eGFP) es van purificar i aïllar correctament però només el producte WT es va utilitzar per els següents experiments estructurals. Malgrat les dificultats de reproduir el que s'havia descrit anteriorment, la proteina finalment es va aconseguir sotmetre a assaijos de funcionalitat. L'assaig de binding es va fer a partir d'una tècnica inusual: voltage-clamp in planar lipid membranes. Aquesta tècnica permet mesurar transport (funció) del transportador sense la necessitat d'incorporarar-la en liposomes. La conclusió final que es pot extreure en aquest treball es que hSGLT1 es pot purificar correctament i que, per tant, obre un gran ventall de possibilitats en un futur, com per exemple, cristalitzar la proteina per tal de resoldre la seva estructura i el seu mecanisme.In the last 20 years, the characterization of membrane protein has become an interesting field for drug design but, the lack of information regarding their mechanism of action makes it even harder to screen for new drug targets. Therefore, studying the structure and mechanism of action of membrane proteins should accelerate the research of new drugs and its medical implications. The project presented here is focused on exploring the features of a human sodium glucose co-transporter (hSGLT1). This transport is involved in absorbing and reabsorbing the glucose in the intestine and it's linked and directly involved in some diseases like: glucose galactose malabsorbation (GGM), diabetes and even some mental diseases like Alzheimer's diseases. The mechanism of action of this transporter is partially understood although some features are still under debate and unclear. However, the 3D structure of this transporter is unknown and it's an end goal for elucidating the mechanism of action of the transporter which will allow screening easily more effective drugs. In order to be available to crystalize a membrane protein for its structure, a huge number of initial steps are necessary. First, the protein of interest must be expressed in a recombinant system satisfactorily and, membrane proteins are not easily expressed in all expression systems. Second, human membrane proteins are even harder to express because it's necessary to work with a non-prokaryote expression system or, otherwise, it is very likely to obtaining in the end a non-functional protein. It has been described, only once, that hSGLT1 could be expressed in a eukaryotic yeast expression system: Pichia pastoris. This yeast has been used a lot in the last 10 years for expressing membrane proteins successfully because, unlike Saccharomyces cerevisae, it can grow much more and therefore obtain more protein in the end. Another advantage is the stability of the expression clones of P. pastoris which eventually leads to a more reproducible result. Although, some disadvantages must be overcome at the same time to have good expression levels like, for example, the clone selection. Because of all of this, the project presented here was initially based on this previous work done in P. pastoris. This thesis is focused on the expression, purification and characterization of hSGLT1 expressed in a yeast heterologous expression system (P.pastoris). Initially, two vectors were design: One expressing hSGLT1 fused to eGFP and another construct without eGFP. The eGFP construct was used to monitorize the protein expression faster and easier which allows optimizing the protein expression by screening for best expression conditions like: induction time, temperature, media and more. Membrane proteins required to be extracted from the membrane in order to purify them and, to do so; a detergent is required. The eGFP fused protein was also used to screen which detergent is more suitable for protein extraction too. Although, both expression product (non-GFP and GFP) were purified and isolated only the WT hSGLT1 was used for further analysis. Despite the difficulties to reproduce what was described before, the protein was eventually subjected to a binding assay. The binding assay was done with an unusual technique which is: voltage-clamp in planar lipid membranes. This technique allowed to measure transport (functionality) of the protein without the need to be incorporated in a liposomes. The main conclusion extracted in this work is that a functional hSGLT1 can be purified which allows a wide number of possibilities in the future like crystallization for elucidating the structure and mechanism of the protein

Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris

Abstract Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes

Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris.

Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes

Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system

Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins. Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K-M,K- but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K-M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake

Hippocampal adaptations in mild cognitive impairment patients are modulated by bilingual language experiences

Bilingualism has been shown to contribute to increased resilience against cognitive aging. One of the key brain structures linked to memory and dementia symptom onset, the hippocampus, has been observed to adapt in response to bilingual experience, at least in healthy individuals. However, in the context of neurodegenerative pathology, it is yet unclear what role previous bilingual experience might have in terms of sustaining integrity of this structure or related behavioral correlates. The present study adds to the limited cohort of research on the effects of bilingualism on neurocognitive outcomes in Mild Cognitive Impairment (MCI) using structural brain data. We investigate whether bilingual language experience (operationalized as language entropy) results in graded neurocognitive adaptations within a cohort of bilinguals diagnosed with MCI. Results reveal a non-linear effect of bilingual language entropy on hippocampal volume, although it does not predict episodic memory performance, nor age of MCI diagnosis

Electrospray ionization of native membrane proteins proceeds via a charge equilibration step

Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion–ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase

Human Albumin Impairs Amyloid β-peptide Fibrillation Through its C-terminus: From docking Modeling to Protection Against Neurotoxicity in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative process characterized by the accumulation of extracellular deposits of amyloid β-peptide (Aβ), which induces neuronal death. Monomeric Aβ is not toxic but tends to aggregate into β-sheets that are neurotoxic. Therefore to prevent or delay AD onset and progression one of the main therapeutic approaches would be to impair Aβ assembly into oligomers and fibrils and to promote disaggregation of the preformed aggregate. Albumin is the most abundant protein in the cerebrospinal fluid and it was reported to bind Aβ impeding its aggregation. In a previous work we identified a 35-residue sequence of clusterin, a well-known protein that binds Aβ, that is highly similar to the C-terminus (CTerm) of albumin. In this work, the docking experiments show that the average binding free energy of the CTerm-Aβ1–42 simulations was significantly lower than that of the clusterin-Aβ1–42 binding, highlighting the possibility that the CTerm retains albumin's binding properties. To validate this observation, we performed in vitro structural analysis of soluble and aggregated 1 μM Aβ1–42 incubated with 5 μM CTerm, equimolar to the albumin concentration in the CSF. Reversed-phase chromatography and electron microscopy analysis demonstrated a reduction of Aβ1–42 aggregates when the CTerm was present. Furthermore, we treated a human neuroblastoma cell line with soluble and aggregated Aβ1–42 incubated with CTerm obtaining a significant protection against Aβ-induced neurotoxicity. These in silico and in vitro data suggest that the albumin CTerm is able to impair Aβ aggregation and to promote disassemble of Aβ aggregates protecting neurons