Biophysical study of the aggregation of the androgen receptor protein in spinal bulbar muscular atrophy

[eng] Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion diseases family; the most famous of which is Huntington disease (HD). SBMA is caused by the expansion of the coding region for the polyQ tract in the exon 1 of androgen receptor (AR), which represents the N-terminal intrinsically disordered transactivation domain (NTD). AR is the nuclear receptor sensible to testosterone and aggregates of this polyQ-expanded protein are observed in the motor neurons of SBMA patients. The aggregation mechanism of polyQ proteins depends both on the length of the tract and on the chemical properties of the regions flanking it, that can increase or decrease the rate of aggregation depending on their secondary structure. In order to study the structure of the polyQ tract in AR and the mechanism by which this protein forms aggregates, we developed recombinant proteins designed over the N-terminal fragment of cleavage of a caspase (Caspase 3) associated to the onset of the toxicity in SBMA. We also developed a set of biophysical tools for rendering these aggregation-prone proteins monomeric and to monitor their evolution from the monomer level to the fibril. These methodologically challenging endeavor allowed us to study the secondary structure of this intrinsically disordered protein as a monomer and then to monitor what regions are important in its oligomerization and aggregation. Bulk biophysical experiments and NMR indicated that the polyQ tract of AR is in α-helical conformation, unlike other polyQ tracts described in literature, and we demonstrated that this conformation is caused by the nucleating effect of an N-terminal flanking sequence of four Leu residues (54LLLL58). We also showed that the helical conformation of this tract prevents the polyQ to acquire the ß-sheet conformation and to progress as a fibril, as a deletion mutant of the 54LLLL58 motif aggregates and forms fibrils faster than the wild type. By measuring the aggregation rates of three different AR recombinant proteins with progressively higher polyQ length (4Q, 25Q and 51Q) emerged that the polyQ is not the only region responsible for oligomerization and we identified by NMR that a second region, N- terminal and far apart from the polyQ is responsible of the early oligomerization. By analysis of the chemical shifts in different NMR experiments we obtained that this region (23FQNLF27) however not entirely helical, is prone to interact and acquire secondary structure. Furthermore, this sequence is known to bind to the ligand binding domain (LBD) of AR in an interaction critical for its dimerization and subsequent translocation into the nucleus, which is called N/C interaction. The crystal structure of this complex shows 23FQNLF27 in α-helical conformation when bound to LBD. We then investigated what amino-acids were important in the interaction stabilizing the intereaction of 23FQNLF27. By mutational analysis and measurements of aggregation rates we demonstrated that the helicity of this region is important for the aggregation and mutations that increase the helicity also an increase the aggregation propensity of the protein. We also identified that the residues responsible for the contact are the Gln in position 2, 28 and 36 which form a ‘spine’ of polar residues in register along the α-helix. This polar side of the helix is not the one in contact with LBD during the N/C interaction and it is possible that the two events occur in parallel. In the complex, we characterized the early oligomerization of AR in the aggregation process associated to SBMA with the perspective to provide valuable information for the development of drugs for this diseases that has currently no treatment.[cat] Les malalties neurodegeneratives són una de les malediccions de la civilització moderna i es troben estretament lligades a l’augment de l’esperança de vida de la població mundial. La majoria d’aquestes malalties estan associades a la deposició de material proteic, altrament conegut com a fibres amiloides, a les neurones i el cervell en general. Les fibres amiloides són conjunts supramoleculars lineals, composats per proteïnes disposades en fulla beta, que mostren una alta rigidesa i estabilitat termodinàmica. Exemples famosos de proteïnes amiloides són la beta amiloide (Aβ), associada a la malaltia d’Alzheimer, i l’α-­‐sinucleïna i la proteïna tau, més estretament lligades a la malaltia de Parkinson. Una altra família de desordres neurodegeneratius associats a la deposició de proteïnes és la de les malalties poliglutamines (poliQ). Aquesta família està formada per nou patologies, entre les que es troben sis atàxies espinocerebrals diferents (de les sigles en anglès, SCA 1, 2, 3, 6, 7, 17), la atròfia dentatorubral-­‐pallidoluysian (de les sigles en anglès, DRPLA) i la atròfia muscular espinal bulbar (de les sigles en anglès, SBMA), històricament la primera en ser descrita. Totes elles són hereditàries, dominants i es manifesten en edat avançada. D’altra banda, totes elles estan associades a l’adquisició de neurotoxicitat degut a l’agregació de la proteïna causant de la malaltia, que s’acumula progressivament a les neurones amb el temps. La mutació responsable de la malaltia és una expansió genètica a la regió polimòrfica de l’ADN que és comuna a totes le proteïnes associades en aquests enfermetats. Aquesta regió polimòrfica és un conjunt de repeticions CAG que codifiquen l’aminoàcid glutamina a nivell d’expressió de proteïna, és per això que es coneix com a tram de poliglutamines. Aquest tram pot tenir diverses longituds, però l’efecte tòxic només té lloc quan es supera un determinat límit d’allargada. Aquest límit fluctua entre 30 i 40 repeticions i varia de malaltia a malaltia, però en tots els casos el número de repeticions influencia la severitat i l’edat en la que s’inicia la malaltia. La raó que explica aquesta inestabilitat genètica resulta de la propensitat de les seqüències d’ADN altament repetitives (com ara els hairpins) que en determina el slippage de la cadena principal durant la replicació de l’ADN. Les expansions més llargues són causades per la reiteració d’aquesta petita mutació i s’ha observat una reducció progressiva de l’estabilitat genètica amb l’increment del número de repeticions, que en última instància determina un avançament temporal i empitjorament dels símptomes. Considerant l’estreta relació entre la presència d’agregats en els teixits dels pacients malalts i l’estadiatge de la malaltia, és fonamental entendre les propietats biofísiques dels trams de poliQ, com aquestes seqüències determinen l’agregació de la proteïna i el tipus d’estructura que presenten els agregats

Fatty acid profile, Desaturase and Atherogenic indices in milk of Holstein Friesian and Italian autochthonous cattle breeds

In the past decades, milk has been considered a mere supplier of nutrients, although its importance was considered paramount for the development and growth of newborns, a number of aspects regarding the biological functions of milk are still unknown. Several positive functional properties of milk derive from fatty acids (FA), mainly unsaturated fatty acids (UFA), either monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids. In particular, UFAs are considered functional components of food because of their positive effects on disease prevention (FAO, 2010; Connor W.E. ,2000;  Wijendran V., 2004). The objective of this study was to characterize the fatty acid profile, the desaturase index and the atherogenic index in milk of local Italian bovine breeds (Cabannina, Varzese and Valdostana) and in a cosmopolitan breed (Holstein Friesian) during the first period of lactation. A total number of 129 cows have been enrolled (Friesian n=30, Cabannina n=30, Varzese n=30, Valdostana n=39) from three dairy farms with similar management and feeding conditions. Animals were chosen in order to have three classes of lactation stage: milk collections were carried out starting from 40±10 days (group A), 70±10 days (group B), and 130±10 days (group C). Milk samples have been analyzed by gas chromatography to obtain the fatty acid profile, on the basis of these results, the Desaturase and Atherogenic Indices were calculated. A   number   of   differences   between   breeds have been  found, in   particular local  breeds showed an higher  percentages  of  UFA,  MUFA,  PUFA,  and  a  higher  UFA/SFA  ratio, as well as lower desaturase indices (related to C14, C16 and C18) and atherogenic index, when compared to Friesian cows. The results can add further information aiming to re-evaluate an almost lost local treasure in Northern Italy.

Satellite SAR interferometric techniques in support to emergency mapping

This paper investigates the potential of Synthetic Aperture Radar (SAR) interferometry in the field of emergency mapping, assessing its suitability for both rapid mapping, aimed at supporting the immediate response phase after a disastrous event, and risk mapping, addressing risk prevention and mitigation activities. The conventional Differential Interferometric SAR technique (DInSAR) and the two currently available multi-temporal interferometric approaches, i.e. Permanent Scatterers (PS) and Small BAseline Subset (SBAS), have been evaluated focusing on the main emergency mapping requirements, namely crisis information product types, availability of optimal input data, requirements in terms of auxiliary data, processing time and expected accuracy. The aforementioned investigations have been carried out exploiting the European Space Agency (ESA) C-band Sentinel-1 mission, characterized by a free, full and open data policy. Therefore, this paper will not assess different SAR sensors and their different technical specifications, e.g. wavelength and space resolution. Representative results are presented and discussed with the aim to describe the possible interferometric product types in specific emergency mapping scenarios

Farmers as data sources: Cooperative framework for mapping soil properties for permanent crops in South Tyrol (Northern Italy)

Abstract Detailed knowledge of agricultural soil properties is a key element for high-quality food production. However, high-resolution soil data covering a large agricultural region are generally unavailable. This study explores a demand-driven cooperative framework for soil data sourcing that connects individual farmers to several stakeholders by means of a centralised database containing more than 16,000 records of soil information collected within the framework of an integrated production program for intensively managed permanent crops in the Adige/Etsch and Venosta/Vinschgau valleys in South Tyrol, Italy. Data for soil pH, soil organic matter (SOM), and soil texture were used to produce digital soil maps with a RMSE of 0.21, 1.25% and a cross-validation of 43%, respectively. Spatialisation was conducted using either regression-kriging or multinomial logistic regression. Collaboration among farmers, public administrators, and researchers provided a successful cooperative framework for digital soil mapping. The maps highlight the complex interplay of the postglacial evolution of these valleys due to the presence of a cluster of large alluvial fans and the anthropogenic influences of intense farming on pH, SOM, and soil texture. This study regarded a subset of the available soil properties, which can be dealt with using the geostatistical approaches presented herein. Thus, a long-term soil monitoring program and the combination of all available variables will allow digital assessment of the spatial patterns of nutrient availability, ecological risk assessments, change detection studies, and an overall long-term plan for soil security at larger spatial scales

Copper and zinc as a window to past agricultural land-use.

Abstract Intensive agricultural management significantly affects soil chemical properties. Such impacts, depending on the intensity of agronomic practices, might persist for several decades. We tested how current soil properties, especially heavy metal concentrations, reflect the land-use history over a 24,000 ha area dominated by intensive apple orchards and viticulture (South Tyrol, ITA). We combined georeferenced soil analyses with land-use maps from 1850 to 2010 in a space-for-time approach to detect the accumulation rates of copper and zinc and understand how present-day soil heavy metal concentrations reflect land-use history. Soils under vineyards since the 1850s showed the highest available copper concentration (median of 314.0 mg kg-1, accumulation rate between 19.4 and 41.3 mg kg-1·10 y-1). Zinc reached the highest concentration in the same land-use type (median of 32.5 mg kg-1, accumulation rate between 1.8 and 4.4 mg kg-1·10 y-1). Using a random forest approach on 44,132 soil samples, we extrapolated land-use history on the permanent crop area of the region, reaching an accuracy of 0.72. This suggests that combining current soil analysis, historical management information, and machine learning models provides a valuable tool to predict land-use history and understand management legacies

Sequence Context Influences the Structure and Aggregation Behavior of a PolyQ Tract

Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets

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

Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor

Polyglutamine (polyQ) tracts are low-complexity regions and their expansion is linked to certain neurodegenerative diseases. Here the authors combine experimental and computational approaches to find that the length of the androgen receptor polyQ tract correlates with its helicity and show that the polyQ helical structure is stabilized by hydrogen bonds between the Gln side chains and main chain carbonyl groups