Дисертації з теми "Calcium sensor proteins"

Calcium plays important roles in both eukaryotic and prokaryotic cells. Its actions help to stabilize cell synthesis, growth and development. In this thesis, studies have been completed to determine effects of calcium and pH on bacterial cell growth and protein expression using the bacterial cell strain E.coli BL21(DE3). Our studies demonstrated the addition of calcium addition in the media does not affect growth but increases protein expression, while reducing the pH from 7 to 4 through the addition of 10mM EGTA in LB media inhibits both. Additionally, we report studies on the design, expression, and purification of fluorescent mCherry variants and their differences in their optical properties, including: extinction coefficients , quantum yields and pKa values. Also, we report progress in the crystallization of two GFP calcium sensors: G1 and D1, using 13 and15% PEG 4000 and 3350 respectively in 50mM HEPES buffer (pH 6.8-7.0) in an effort to optimize crystallization.

Monitoring Ca²⁺ in intracellular compartments remains an uphill task. Fluorescent, organic calcium dyes and genetically encoded calcium indicators (GECIs) have been widely used to increase our understanding of neural activity. Notable among the GECIs is the GCaMP family. The first versions of this family showed potential but were relatively dim and had poor signal to noise ratios. To overcome these weaknesses, our laboratory developed three new ratiometric versions of the SyGCaMP2 family called SyGCaMP2-mCherry, SyGCaMP2-mCherry⁺ and GCaMP2-mCherry. The purpose of the work presented here was to establish whether these sensors were functional and to then use them to examine calcium signalling within the presynaptic terminals of cultured hippocampal and cerebral cortical neurones. The sensors were expressed in HEK 293T cells and their sensitivities to changes in intracellular free Ca²⁺concentration ascertained. The dissociation constants calculated for SyGCaMP2-mCherry⁺ and SyGCaMP2-mCherry were 140 ± 3.1 nM and 149 ± 3.3 nM respectively, but GCaMP2-mCherry was not accurately measured. Sensors were expressed in cortical and hippocampal neurones using lipofection based transfection methods and the expression patterns of each recorded. Both SyGCaMP2-mCherry and SyGCaMP2-mCherry⁺ were expressed in puncta that co-localised with presynaptic markers bassoon and VGlut1. Neurones were activated using field stimulation and the responses to different intensities and patterns of stimulation evaluated. Using the Thy1.2 promotor for neuronal expression, two transgenic mice (SyG 14 and SyG 37) were engineered in our lab that expressed SyGCaMP2-mCherry and responses to electrical stimulation were characterised in hippocampal brain slices. The roles of intracellular Ca²⁺stores in shaping the presynaptic Ca²⁺dynamics were examined and results demonstrated that inhibiting the sarcoendoplasmic reticulum calcium transport ATPase (SERCA pump) with Cyclopiazonic acid (CPA) and thapsigargin led to an enhancement in synaptic strength. In addition, activating ryanodine receptors (RYRs) with caffeine and low concentrations of ryanodine altered presynaptic Ca²⁺ dynamics.

In this study the mechanisms of Ca²⁺-induced-Ca²⁺-release, effects of membrane depolarizations and the actions of pharmacological intracellular Ca²⁺-modulators were examined in cultured rat dorsal root ganglion (DRG) neurones. The whole cell configuration of the patch clamp technique was used to record action potentials, action potential after-potentials and voltage-activated calcium currents, (I_Ca), calcium-activated chloride currents, (I_CI(Ca)), and non-selective cation currents, (I_CAN), under current and voltage clamp recording conditions, respectively. A sub population of DRG neurones expressed action potential after-depolarizations and I_CI(Ca) tail currents which were due to activation of Ca²⁺-activated Cl^- channels as a result of Ca²⁺ entry. I_CAN was dominantly activated due to Ca²⁺ release from intracellular stores evoked by pharmacological Ca²⁺-releasing agents such as caffeine, ryanodine and dihydrosphingosine. Calcium-activated conductances were identified by estimating reversal potentials of the activated currents, using selective pharmacological blockers and extracellular ionic replacement studies. Calcium-dependence of activated currents was also examined by using high concentration of intracellular Ca²⁺ buffer, EGTA, to prevent elevation of intracellular Ca²⁺-levels and by rapidly buffering raised intracellular Ca²⁺ using intracellular 'caged Ca²⁺ chelator', diazo-2. The involvement of intracellular Ca²⁺- stores was examined by performing experiments in Ca²⁺-free extracellular recording medium and pharmacologically inhibiting release of Ca²⁺ from intracellular stores, using dantrolene. Ryanodine had complex actions on DRG neurones, which reflected its ability to mobilize Ca²⁺, deplete Ca²⁺ stores, and inhibit Ca²⁺ release channels. Ryanodine inhibited action potential after-depolarizations and I_CI(Ca) tail currents by interacting with intracellular stores and preventing amplification of Ca²⁺ signalling by CICR. It was found that CICR observed under physiological conditions in rat DRG neurones involves intracellular Ca²⁺ stores which were sensitive to ryanodine. In addition to ryanodine sensitivity these intracellular Ca²⁺ stores could be mobilized by caffeine and dihydrosphingosine.

Hippocalcin-like protein 1 (HPCAL1) is a neuronal calcium sensor (NCS) protein found in the brain and in the retina. NCS proteins have important roles in signaling pathways including phototransduction; however, the role of HPCAL1 in the retina remains unresolved. The objective of this thesis is to characterize HPCAL1 and identify its potential interacting partners in the retina. The first part of the thesis examines the localization and characteristics of HPCAL1 using a variety of biochemical assays. The presence of HPCAL1 in the retina was confirmed with RT-PCR, Western blotting analysis, and immunofluorescence microscopy. Since NCS proteins respond to intracellular calcium level changes, HPCAL1 was expressed and isolated from both mammalian cells and E. coli to study its calcium binding properties and other characteristics. Results from a gel mobility shift assay and a fluorescence assay clearly indicated that HPCAL1 undergoes conformational changes upon calcium binding. Furthermore, membrane association assays confirmed that retinal HPCAL1 possesses the calcium-myristoyl switch mechanism which responds to the presence or absence of calcium. NCS proteins often interact with other proteins to perform their functions; therefore, the second part of the study involves the use of mass spectrometry in an attempt to identify calcium-dependent interacting partners of HPCAL1. Over 300 potential interacting partners were identified, and selected proteins were subjected to co-immunoprecipitation and Western blotting analysis or co-localization studies using immunofluorescence microscopy in order to confirm their interactions with HPCAL1. TorsinA has been identified as a calcium-dependent interacting partner to HPCAL1; however, further studies will have to be conducted to determine the significance of this interaction and to confirm other potential interacting partners that were identified in the mass spectrometry analysis. The results of this study provide important technical information on the biochemical characterization of HPCAL1 and the properties of HPCAL1 in the retina. Not only has the identification of potential interacting partners of HPCAL1 provided first insights at its possible role or function in the retina, it has also pointed out the potential for identifying interacting partners of other NCS proteins using mass spectrometry.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic activity in various cellular compartments are highly desired. In this dissertation, we report a novel strategy to create protease sensors by grafting an enzymatic cleavage linker into a sensitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage, which can be used to determine protease activity and track protease activation and inhibition with a ratiometric measurement mode in living cells. Our designed protease sensors exhibit large relative ratiometric optical signal change in both absorbance and fluorescence, and fast response to proteases. Meanwhile, these protease sensors exhibiting high enzymatic selectivity and kinetic responses are comparable or better than current small peptide probes and FRET-based protease probes. Additionally, our protease sensors can be utilized for real-time monitoring of cellular enzymogen activation and effects of inhibitors in living cells. This novel strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe disease mechanisms corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects. Strong fluorescence was still retained in the cleaved EGFP-based protease sensors, which stimulated us to identify the EGFP fragment with fluorescence properties for further understanding chromophore formation mechanisms and investigating protein-protein interactions through fluorescence complementation of split EGFP fragments. Through fusing EF-hand motifs from calbindin D9k to split EGFP fragments, a novel molecular probe was developed to simultaneously track the calcium change or calcium signaling pathways and calcium-dependent protein-protein interaction in living cells in real time.

Thesis (Ph. D.)--Georgia State University, 2008.
Title from title page (Digital Archive@GSU, viewed July 1, 2010) Jenny J. Yang, committee chair; Edward Brown, Giovanni Gadda, Zhi-ren Liu, committee members. Includes bibliographical references (p. 230-258).

My scientific interest is focused on the field of cellular electrical activity, ranging from the study of intracellular enzymatic processes to the characterization of new generation of drugs. For this purpose I also used the most powerful techniques of investigation, including patch-clamp technique, fluorescence imaging, and surface plasmon resonance (SPR) spectroscopy. Moreover, to shed light on complex molecular mechanisms, unconventional strategies were employed, requiring sometimes the realization of specific devices not commercially available. In particular my PhD thesis includes two different scientific projects: the biophysical characterization of antimicrobial peptides and the modulation of visual phototransduction in vertebrate cones. In the first project, the patch-clamp technique was employed to study the pore forming properties of synthetic cecropin-melittin hybrid peptide (CM15), alamethicin F50/5 and its synthetic analog [L-Glu(OMe)7,18,19] under strict physiological conditions. These short peptides selectively permeabilize the bacteria plasma membrane leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. I pursued this study by recording the ion current through the channels formed by these peptides, once inserted in the membrane of photoreceptor rod outer segment membrane (OS) isolated from frog retinae. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained, it was demonstrated that, different than alamethicins, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate that CM15 form pores according to a toroidal model. Moreover, in order to understand the divalent-cation dependency of [L-Glu(OMe)7,18,19] binding to the lipid bilayer at the molecular level, the electrophysiological experiments were paralleled with experiments employing SPR spectroscopy. Results indicate the presence of Ca2+ in the external solution increases the probability of formation of smaller and more stable [L-Glu(OMe)7,18,19] pores. The second project of this thesis concerns the investigation of the physiological role of the neuronal calcium sensor zGCAP3 in the photrasduction cascade. This study was pursued through the simulation of an over expression and a knock-down of this protein, by delivering it, or its monoclonal antibody, into zebrafish cone cytosol, while recording their photoresponses with the patch-clamp technique. The intracellular protein delivery was attained via the patch pipette, by ejecting the proteins out of a tube inserted into the pipette lumen. A microperfusion system was employed to apply the desired exogenous molecules with a precise timing. However, the long tapered shape of the pipette shank make it very difficult to perfuse efficiently the cell with this strategy. For this reason a pressure polishing setup was assembled to enlarge the patch pipette shank, through the calibrated combination of heat and air pressure. This allowed to insert quartz or plastic tubes in the pipette lumen very close to its tip. In order to obtain a substantial and specific silencing of the zGCAP3s in zebrafish cones, surface plasmon resonance experiments were performed to allow the selection of a monoclonal antibody with strong affinity for zGCAP3 and low cross interaction with other components of the phototransduction cascade. Results showed that the perfusion with GCAP3 did not altered significantly the light response, while he anti-zGCAP3 incorporation in the cytosol caused the progressive photoresponse fall, followed by the progressive fall of saturating flash response amplitude, probably due to the progressive GC inhibition. The unexpected lack of an effect of zGCAP3 incorporation in the cone cytosol, suggests that the endogenous number of zGCAP3 is saturating, i.e. their number is equal or above the number of their target molecules (guanylate cyclase), therefore any further increase of zGAP3 in the cytosol is uneffective.

[EN] Persimmon (Diospyros kaki L.) 'Rojo Brillante' is an astringent variety characterised by good growing conditions, excellent colour, size, sensory characteristics and good nutritional properties. In the last decade, its production has grown substantially in Spain given the application of high levels of CO2 to remove astringency while firmness is preserved. This technology has also increased its potential as a fresh-cut commodity. However, physical damage during processing result in degradation of the colour and firmness of the product and a higher susceptibility to microbial spoilage that significantly reduces the fruit's shelf life. The objective of the present thesis was to develop optimum procedures for processing and marketing 'Rojo Brillante' persimmon into a fresh-cut product with the maximum shelf life and best physicochemical, nutritional, sensory and microbiological quality. Firstly, the objective was to evaluate the effect of the maturity stage (MS) at harvest, storage time at 15 ºC before processing, and the application of different antioxidant treatments on enzymatic browning, sensory and nutritional quality of fresh-cut 'Rojo Brillante' persimmon during storage at 5 ºC. Concentrations of 10 g L-1 ascorbic acid (AA) or 10 g L-1 citric acid (CA) controlled tissue browning and maintained the visual quality of fresh-cut persimmon above the limit of marketability for 6-8 storage days at 5 ºC, depending on the MS. However, these acidic solutions reduced fruit firmness as compared to control samples. Further studies showed that the combination of these antioxidants with 10 g L-1 CaCl2 maintained firmness of the persimmon slices within the same range as the control samples. In another work, the application of 1-methylcyclopropene (1-MCP) allowed to process fruits after 45 days of storage at 1 ºC with commercial firmness and the antioxidant solution (10 g L-1 CA + 10 g L-1 CaCl2) extended the limit of marketability up to 9 days of storage at 5 ºC. Different controlled atmosphere conditions in combination with AA or CA dips were also evaluated as a first step to select optimum O2 and CO2 concentrations for modified atmosphere packaging (MAP) of fresh-cut 'Rojo Brillante' persimmons. Overall, the combination of antioxidant dips and a controlled atmosphere composed of 5 kPa O2 (balance N2) was proved to be the most effective combination to control enzymatic browning. This atmosphere maintained the visual quality of persimmon slices within the limit of marketability during 7- 9 days at 5 ºC. On the contrary, high CO2 concentrations (10 or 20 kPa) induced darkening in some tissue areas, associated with a flesh disorder known as 'internal flesh browning'. Later studies confirmed the beneficial effect of an active MAP in 5 kPa O2 compared to passive MAP to improve the visual quality of fresh-cut 'Rojo Brillante' persimmon, showing a synergic effect with the antioxidant dip (10 g L-1 CA + 10 g L-1 CaCl2). Antioxidant edible coatings were prepared from whey protein isolate (WPI), soy protein isolate (SPI), hydroxylpropyl methylcellulose (HPMC) and apple pectin as the polymeric matrix. All edible coatings were amended with the antioxidant combination selected (10 g L-1 CA + 10 g L-1 CaCl2). All the edible coatings tested proved effective to control enzymatic browning of persimmon slices. However, the samples treated with the HPMC- and pectin- based coatings were scored with a better visual quality that the rest of the treatments. In general, free radical scavenging activity and total carotenoid content increased in late-season persimmons; whereas, processing (cutting and storage at 5 ºC), antioxidant dips, controlled atmosphere storage or edible coatings had no clear effect on nutritional quality (vitamin C, free radical scavenging activity, total phenolic content, and carotenoids) of fresh-cut persimmons.
[ES] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' es un cultivar astringente que presenta unas propiedades organolépticas y nutricionales excelentes. En la última década, su cultivo en el área mediterránea de España se ha incrementado de manera exponencial con el desarrollo de la tecnología que permite eliminar la astringencia, manteniendo la firmeza del mismo. Esta nueva forma de presentación, aporta numerosas ventajas, entre la que se incluye la posibilidad de ser comercializado como fruta fresca cortada. Sin embargo, el éxito comercial del producto está limitado por el pardeamiento enzimático, la pérdida de firmeza y al crecimiento microbiano. En este contexto, el objetivo de la Tesis ha sido el desarrollo de caqui 'Rojo Brillante' fresco cortado mediante un enfoque que integra el estudio de las características del producto en el momento del procesado y de distintas tecnologías que mantengan la calidad físico-química, sensorial, nutricional y microbiológica del producto durante un periodo que permita su comercialización. En primer lugar, se evaluó el efecto del estado de madurez (MS) en el momento de recolección, el tiempo de almacenamiento a 15 ºC antes del procesado y la aplicación de diferentes antioxidantes en el pardeamiento enzimático y la calidad sensorial y nutricional del caqui 'Rojo Brillante' cortado y almacenado a 5 ºC. La aplicación de 10 g L-1 de ácido ascórbico (AA) ó 10 g L-1 ácido cítrico (CA) controló el pardeamiento enzimático y mantuvo la calidad visual del caqui por encima del límite de comercialización entre 6 y 8 días de almacenamiento a 5 ºC, dependiendo del MS. Sin embrago, la aplicación de estos antioxidantes redujo de manera significativa la firmeza del fruto respecto al control. La combinación de estos antioxidantes con 10 g L-1 de CaCl2 permitió mantener la firmeza en el mismo rango que las muestras control. En un trabajo posterior, la aplicación de 1-metilciclopropeno (1-MCP) permitió procesar caqui almacenado 45 días a 1 ºC con una buena firmeza comercial y el tratamiento antioxidante (10 g L-1 CA + 10 g L-1 CaCl2) consiguió alcanzar un límite de comercialización del producto de 9 días a 5 ºC. La evaluación de distintas atmósferas controladas en combinación con tratamientos antioxidantes (AA o CA), como paso previo al envasado en atmósfera modificada (MAP) del caqui, mostró como más efectiva en el control del pardeamiento enzimático la atmósfera compuesta por 5 kPa O2 (balance N2). Esta atmósfera mantuvo la calidad visual del caqui cortado dentro del límite de comercialización durante 7-9 días a 5 ºC. Por el contrario, la aplicación de altas concentraciones de CO2 (10 ó 20 kPa) dio lugar a un pardeamiento en ciertas zonas de la pulpa que se conoce como 'internal flesh browning'. Estudios posteriores confirmaron el efecto beneficioso del envasado de caqui cortado y tratado con solución antioxidante (CA-CaCl2) en una MAP activa de 5 kPa O2 en la calidad visual del fruto frente a la aplicación de una MAP pasiva. El desarrollo de recubrimientos comestibles con capacidad antioxidante se realizó mediante la incorporación de antioxidantes (10 g L-1 CA + 10 g L-1 CaCl2) a formulaciones a base de proteína de suero lácteo (WPI), proteína de soja (SPI), hidroxipropilmetilcelulosa (HPMC) y pectina. Todos los recubrimientos fueron efectivos controlando el pardeamiento enzimático del caqui cortado, siendo las muestras recubiertas con HPMC y pectina las mejor evaluadas visualmente. En general, el procesado, la aplicación de antioxidantes, el envasado en atmósferas controladas y los distintos recubrimientos comestibles estudiados, si bien no mostraron un efecto claro en los parámetros de calidad nutricional evaluados, no tuvieron un efecto negativo en los mismos. Por otra parte, los frutos cosechados a final de campaña tuvieron mayor actividad antioxidante y contenido en carotenoides.
[CAT] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' és un cultiu astringent que presenta unes propietats organolèptiques i nutricionals excel¿lents. En la última dècada, el seu cultiu en l'àrea mediterrània d'Espanya s'ha incrementat de manera exponencial amb el desenvolupament de la tecnologia que permet eliminar l'astringència, mantenint la fermesa del mateix. Esta nova forma de presentació, aporta un gran nombre d'avantatges, entre els quals s'inclou la possibilitat de comercialitzar-lo com fruita fresca processada. No obstant, l'èxit comercial del producte està limitat per pardetjament enzimàtic, la pèrdua de fermesa i el creixement microbià. L'objectiu de la Tesis ha estat en el desenvolupament de caqui 'Rojo Brillante' tallat en fresc mitjançant un enfocament que integra l'estudi de les característiques del producte en el moment del processat i de diferents tecnologies en el manteniment de la qualitat físico-química, sensorial, nutricional i microbiològica del producte durant un període que permeta la seua comercialització. En primer lloc, es va avaluar l'efecte de l'estat de maduresa (MS) en el moment de recol¿lecció, el temps d'emmagatzemament a 15ºC abans del processat i l'aplicació de diferents tractaments antioxidants en el pardetjament enzimàtic i la qualitat sensorial i nutricional del caqui 'Rojo Brillante' tallat i emmagatzemat a 5 ºC. L'aplicació de 10 g L-1 d'àcid ascòrbic (AA) o 10 g L-1 d'àcid cítric (CA) va controlar el pardetjament enzimàtic i va mantenir la qualitat visual del caqui per damunt del límit de comercialització entre 6-8 dies d'emmagatzemament a 5 ºC, depenent del MS. No obstant, l'aplicació d'antioxidants va reduir de manera significativa la fermesa del fruit comparat amb el control. La combinació d'aquestos antioxidants amb 10 g L-1 de CaCl2 va permetre mantenir la fermesa en el mateix rang que les mostres control. En un treball posterior, l'aplicació de 1-metilciclopropeno (1-MCP) va permetre processar caqui emmagatzemat 45 dies a 1 ºC amb una bona fermesa comercial i a més, el tractament antioxidant (10 g L-1 CA + 10 g L-1 CaCl2) va aconseguir un límit de comercialització del producte tallat de 9 dies a 5 ºC. L'avaluació de diferents atmosferes controlades en combinació amb tractaments antioxidants (AA o CA), com a pas previ a l'envasament en atmosfera modificada (MAP) del caqui 'Rojo Brillante, va mostrar com a més efectiva en el control del pardetjament enzimàtic l'atmosfera composta per 5 kPa O2 (balanç N2). Aquesta atmosfera va mantenir la qualitat visual del caqui tallat dins del límit de comercialització durant 7-9 dies a 5 ºC. Per contra, l'aplicació d'altes concentracions de CO2 (10 ó 20 kPa) va donar lloc a un pardetjament en certes zones de la polpa, el qual és conegut com 'internal flesh browning'. Estudis posteriors van confirmar l'efecte beneficiós de l'envasament de caqui tallat i tractat amb solució antioxidant (CA-CaCl2) en una MAP activa de 5 kPa O2 millorant la qualitat visual de la fruita front a l'aplicació de una MAP passiva. El desenvolupament de recobriments comestibles amb capacitat antioxidant es va realitzar mitjançant la incorporació d'antioxidants (CA-CaCl2) en formulacions a base de proteïna de sèrum làctic (WPI), proteïna de soia (SPI), hidroxipropilmetilcel-lulosa (HPMC) i pectina. Tots els recobriments van ser efectius controlant el pardetjament enzimàtic del caqui tallat. No obstant, les mostres recobertes amb HPMC i pectina van ser millor avaluades visualment que la resta de tractaments. En general, el processat, l'aplicació d'antioxidants, l'envasament en atmosferes controlades i els distints recobriments comestibles estudiats, si bé no van mostrar un efecte clar en els paràmetres de la qualitat nutricional avaluats, no van tindre un efecte negatiu en els mateixos. Per altra banda, els fruits recol¿lectats a final de temporada van tenir major activitat antioxidant i contingut en
Sanchís Soler, E. (2016). Effect of processing on the physicochemical, sensory, nutritional and microbiological quality of fresh-cut 'Rojo Brillante' persimmon [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62588
TESIS

Proteins are biomolecules involved in virtually every process occurring in cells, characterized by a sequence of amino acids, which confer them peculiar structural features implying specific functions. Ca2+-sensor proteins are a family of proteins whose function is determined by structural changes occurring upon Ca2+-binding, taking part in a wide range of physiological and pathological processes, among which muscle contraction and phototransduction. Unraveling the complex machinery behind these physiological processes is fundamental not only for the sake of expanding our knowledge, but also to understand which mechanisms are altered in inherited pathological conditions, in order to develop potential therapeutic approaches that may help relieving or treating these disorders. Protein therapy is one of the most promising potential treatments, allowing for the substitution of dysfunctional protein pool with physiological variants. Here, a collection of biochemical and biophysical studies focused on the physiological and pathological aspects of some Ca2+-sensor proteins is presented, together with possible therapeutic implications in nanomedicine using CaF2 nanoparticles (NP) as protein carriers. The combination of experimental and computational techniques revealed itself a complementary and exhaustive approach, allowing for a multiscale investigation of protein structural and functional properties. Indeed, the high-resolution structural information provided by Molecular Dynamics (MD) simulations and Protein Structure Network (PSN) analysis can be proficiently integrated into the biophysical and biochemical experimental framework constituted by Circular Dichroism (CD) and fluorescence spectroscopy, Dynamic Light Scattering (DLS), Ca2+-binding assays and enzymatic assays. Such integrated approaches allowed us to discover that mutations of the Neuronal Calcium Sensor (NCS) protein Guanylate Cyclase Activating Protein 1 (GCAP1) associated to retinal dystrophies did not necessarily altered protein Ca2+-affinity, but rather that the pathological dysregulation of the target enzyme Guanylate Cyclase (GC) depended on more complex mechanisms, probably involving modified intramolecular communication. Moreover, PSN analysis of MD trajectories of GCAP1 highlighted that small conformational variations may strongly impact protein functionality, as the switch between activator and inhibiting states occurs through minor structural rearrangements subsequent to the Ca2+/Mg2+ exchange in specific binding sites. This suggested again that the investigation of intramolecular communication may be the key to clarify unknown complex mechanisms of not only of Ca2+-sensor proteins, but also of proteins belonging to different superfamilies.

Calcium is a cation which plays a pivotal role as second messenger, thus its concentration in cells needs to be finely regulated. Many systems work for that purpose, including Ca2+ sensor proteins, which undergo conformational changes upon Ca2+ coordination via EF-hands. Ca2+ sensors can be ubiquitous or tissue specific. Examples in this sense are represented by Guanylate Cyclase Activating Protein 1 (GCAP1) and Calcium- and Integrin-Binding Protein 2 (CIB2), involved in sight and hearing respectively. Missense point mutations in GCAP1 and CIB2 were found to be associated with genetic diseases characterized by retinal dystrophies and/or deafness. During my PhD, I focused my attention on the characterization of two point mutations namely p.Glu111Val (E111V) in GCAP1, leading to Cone/Rod dystrophy in an Italian family, and p.Glu64Asp (E64D) in CIB2, linked to Usher syndrome type 1J (USH1J), a rare disease characterized by the copresence of blindness and deafness. In particular, I spent the first part of the PhD investigating the role of CIB2 which is still under debate, finding that it is per se uncapable to work as a Ca2+ sensor under physiological conditions and that the conservative mutation linked to USH1J perturbs an allosteric communication between pseudo-EF1 and EF3, thus blocking the protein in an unfunctional conformation. Then, I characterized E111V GCAP1, finding that it is incapable of regulating its molecular target (Guanylate Cyclase), leading to a constitutive active enzyme and thus a progressively high concentrations of Ca2+ and cGMP in cells, which may explain the pathological phenotype. Looking for a potential therapeutic approach for Cone-Rod dystrophies, we found that the well-established Ca2+-relay model, explaining the gradual activation of Guanylate Cyclase by multiple GCAP molecules following gradual changes in intracellular Ca2+ concentrations, seems to be species-specific, since it apparently does not work in the same way in humans as in mouse and bovine photoreceptors. Finally, we identified a general method for the characterization of the interaction between a ubiquitous Ca2+ sensor protein (calmodulin) and inorganic CaF2 nanoparticles, suggesting their suitability as devices for nanomedicine applications.

Eyes are responsible for detection of light in the retina and the conversion of a light stimulus into an electrical signal that is ultimately conveyed to the visual cortex of the brain. The first step of vision occurs during the phototransduction cascade, which takes place in specialized cells in the retina, namely rods and cones. In the complex mechanism of phototransduction, a cascade of biochemical events is regulated by variations of the concentration of two second messengers, cGMP and calcium. Their intracellular concentrations are strictly interconnected and finely regulated by guanylate cyclase-activating proteins (GCAPs). GCAPs are neuronal calcium sensor proteins that detect subtle variations of calcium concentration and thereby regulate the enzymatic activity of membrane-bound Guanylate Cyclases (GCs), that synthetize cGMP, through a Ca2+- dependent feedback mechanism. Three isoforms of GCAPs have been described in the human retina (GCAP1, 2 and 3) with a different distribution in the photoreceptor cells; however, the physiological meaning of this apparent redundancy is not well understood. The best-known human isoform is the GCAP1, whereas no thorough biochemical investigation of the molecular proprieties of human GCAP2 and GCAP3 has been performed so far. My PhD research is focused on the biochemical and biophysical characterization of human GCAP2 and GCAP3 with the goal to understand their structural properties and their role in the phototransduction cascade. Beside focusing on their physiological role, I also investigated the pathological determinants of these two proteins by evaluating the effects of single point mutations in their associated variant, G157R-GCAP2 and D101H-GCAP3 respectively, which have been associated with inherited retinal diseases. A promising approach for treating these genetic disorders is the use of liposomes as potential delivery system for therapeutic molecules, such as functional proteins or genes. To probe the potential of liposomes as a tool to replace mutated variants with functional and active proteins, initial experiments have been performed with cultured HEK293 cells, with the aim to understand the mechanism of liposomes up-take and the release of physiologically active biomolecules, such as DNA and functional GCAP. A common disease that affects vision, with fare less harmful effects compared to retinal dystrophies, is Dry Eye disease (DED). A key role in DED is played by mucins. These are glycoproteins secreted by conjunctival goblet cells that are responsible to create a stable tear film and to protect and maintain the integrity of the ocular surface. It has been reported that the gene expression of mucins, and in particular mucin-4, were significantly lower in conjunctival epithelium of patients affected by DED and altered levels of these glycoproteins could be restored by the topical administration of eyedrops. In the second part of my PhD I investigated the effects of the treatment with hyaluronic acid cross-linked with urea (U-HACL) eyedrop and characterized the expression levels of mucins before and after treatment in patients affected by DED.

Calcium (Ca2+) is an ubiquitous key second messenger in all living organisms, from bacteria to differentiated neuronal cells and is involved in a large variety of biological processes such as apoptosis, cell differentiation and proliferation, fertilization, transcription factors activation and many others. Plants in particular are known to be capable of exploiting Ca2+ signalling in a more sophisticated way than other organisms as demonstrated by the larger set of Ca2+-binding proteins (CBPs). Although the study of CBP has been an open and highly studied field for over a century, a biochemical and structural characterization of many CBPs from different organisms is still missing. Here, we provided a deep characterisation of two plant (Arabidopsis thaliana) Ca2+ sensors, a specific class of CBPs, to understand the differences in their ability to respond to Ca2+ and the regulation of downstream targets. Thus we analysed the metal and target binding properties of Calmodulin like protein 7 and 19 (CML7 and CML19). By exploiting spectroscopic techniques (NMR and CD) we evaluated the conformational changes induced by metal binding and assigned the specific affinities for each binding site by a combination of calorimetric and spectroscopic techniques (ITC and NMR). To further understand the Ca2+ sensing properties and the ability to interact with target proteins, we analysed the interaction of CML7 and CML19 with synthetic peptides, representing the binding region on physiological or model targets. Because the majority of CBPs are characterised by multiple metal binding sites, clustered affinities and cooperativity events, the determination of Ca2+/CBPs stoichiometry requires time and sample consuming procedures and sophisticated instrumentations (i.e. ITC and NMR). Thus, we report the optimisation of a new method, based on X-ray fluorescence, to overcome the most common limitations of the currently used techniques, allowing the quick estimation of the Ca2+/protein stoichiometry using low amounts of sample. Finally, initial studies on the recombinant production and characterisation of a neuronal family of CBPs are reported. These protein are called NECABs and are characterised by a peculiar domain composition and might be involved in the development of neurodegenerative diseases.

Nelle piante Il calcio (Ca2+) regola una vasta gamma di risposte fisiologiche agli stimoli esterni. Oltre alla Calmodulina (CaM), altamente conservata, le piante possiedono una famiglia di 50 proteine calmodulin-like (CMLs), presente soltanto in esse, che gioca un ruolo importante nello sviluppo e nella risposta agli stress. Tuttavia, ad oggi, sono state determinate sperimentalmente le proprietà di poche CMLs. Il presente studio riporta una dettagliata caratterizzazione biochimica e biofisica in vitro di due proteine calmodulin-like ricombinanti, CML14 e CML36 di Arabidopsis thaliana, con lo scopo di chiarire se queste proteine abbiano le caratteristiche tipiche dei Ca2+ sensori. La proteina CML14 possiede tre motivi EF-hands di cui uno solo è predetto essere un sito di legame funzionale per il Ca2+, mentre nonostante la presenza di quattro motivi EF-hands, soltanto 3 di essi sono predetti essere funzionali nelle proteina CML36. Per analizzare la forza di legame del Ca2+ e del magnesio (Mg2+) alle proteine CML14 e CML36 abbiamo sfruttato la calorimetria di titolazione isotermica (ITC). Per valutare gli effetti del legame dei metalli sulla struttura e sui cambiamenti conformazionali delle due proteine abbiamo sfruttato la spettroscopia di risonanza magnetica nucleare (NMR), il dicroismo circolare (CD), la cromatografia di esclusione molecolare (SEC) e la fluorescenza sia intrinseca che basata su un fluoroforo estrinseco (ANS). Inoltre, per studiare la stabilità termica e locale di entrambe le proteine abbiamo sfruttato la calorimetria differenziale a scansione (DSC) e la proteolisi limitata. I nostri risultati hanno dimostrato che la proteina CML14 lega un solo ione Ca2+ con una affinità dell’ordine di micromolare (Kd ~ 12 µM), mentre la presenza del Mg2+ diminuisce l’affinità della proteina per il Ca2+ di circa 5 volte. Sebbene il legame del Ca2+ alla proteina aumenta la stabilità termica e locale, esso non induce né l’aumento dell’idrofobicità a livello della superficie della proteina né un grande riarrangiamento conformazionale. Tuttavia, Il legame del Ca2+ causa cambiamenti strutturali localizzati nell’unico EF-hand funzionale. Per quanto riguarda la proteina CML36, i nostri dati hanno rivelato che il Mg2+ e il Ca2+ inducono cambiamenti conformazionali della proteina significativi e differenti. In particolare, il legame del Ca2+ alla proteina aumenta la stabilità locale e in linea con una funzione di Ca2+ sensore, la proteina va incontro ad un aumento dell’idrofobicità di superficie in maniera dipendente dal Ca2+ ma non dal Mg2+. Inoltre, i nostri risultati ci hanno permesso di speculare che la proteina CML36 nella forma apo- è nello stato flessibile di molten globule. La proteina CML36 lega tre ioni Ca2+ in assenza di Mg2+ con un sito di legame ad alta affinità (Kd ̴ 0.7 µM) e due siti a bassa affinità (Kd ̴ 47 µM), e due ioni Ca2+ in presenza di Mg2+ (Kd1 ̴ 0,7 µM e Kd2 ̴ 5 µM). Questi dati suggeriscono che, in condizioni fisiologiche, Il Mg2+ è legato in maniera costitutiva ad un EF-hand mentre il Ca2+ si associa funzionalmente soltanto a due EF-hands. Considerati nell’insieme, i nostri risultati suggeriscono che la proteina CML14 non sembra avere le caratteristiche di un Ca2+ sensore classico e, in contrasto con una funzione controllata dall’esposizione delle regioni idrofobiche sulla superficie della proteina, indicano un ruolo diverso del Ca2+ nell’interazione di essa con un possibile target. Contrariamente la proteina CML36 mostra le caratteristiche tipiche di un Ca2+ sensore. In conclusione il nostro lavoro fornisce informazioni nuove ed interessanti sulle proprietà biochimiche delle proteine CML14 e CML36 di Arabidopsis che possono essere utilizzate per studi futuri allo scopo di chiarire il loro ruolo fisiologico preciso.
Calcium (Ca2+) regulates a wide range of physiological responses to external stimuli in plants. Besides conserved Calmodulins (CaMs), plants possess a unique family of 50 calmodulin-like proteins (CMLs) playing important roles in plant development and stress responses. However, to date, only the Ca2+ binding properties of few CMLs have been experimentally determined. The present study reports a detailed biochemical and biophysical in vitro characterization of two recombinant CMLs, namely, CML14 and CML36 from Arabidopsis thaliana, undertaken in order to clarify whether these proteins may be endowed with the typical features of the Ca2+ sensors. CML14 possesses three EF-hand motifs, but only one is predicted to be a functional Ca2+ binding site, whereas CML36 is predicted to have three functional Ca2+ binding sites despite the presence of four EF-hand motifs. We applied isothermal titration calorimetry (ITC) to analyse the energetics of Ca2+ and Mg2+ binding to CML14 and CML36. In addition nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy, size exclusion chromatography (SEC) were used together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry (DSC) and limited proteolysis were used to characterize proteins thermal and local stability. Our results demonstrated that CML14 binds one Ca2+ ion with a micromolar affinity (Kd ~ 12 µM) while, the presence of Mg2+ decreases the Ca2+ affinity by ~ 5-fold. Although binding of Ca2+ to CML14 increased thermal and local stability, it does not result in a more hydrophobic protein surface and does not induce a large conformational rearrangement. However Ca2+ binding causes only localized structural changes in the unique functional EF-hand. Regarding the CML36 protein, our data revealed that the binding of Mg2+ and Ca2+ induce significant and distinct conformational changes throughout the protein. In particular, the binding of Ca2+ to CML36 increased the local stability and in line with a Ca2+ sensor function, the protein underwent a Ca2+- dependent increase in surface-exposed hydrophobicity while Mg2+ dependent effect was not noted. In addition, our findings enabled us to speculate on the apo- form of CML36 as a flexible molten globule state. CML36 binds three Ca2+ ions in the absence of Mg2+ with a high affinity binding site (Kd ~ 0.7 µM) and two lower affinity sites (Kd ~ 47 µM), and two Ca2+ ions in the presence of Mg2+ (Kd1 ~ 0.7 µM and Kd2 ~ 5 µM). These data suggest that, under physiological conditions, one EF-hand must be constitutively bound to Mg2+ while Ca2+ binds functionally only to two EF-hands. Together our results suggested that CML14 does not appear to be endowed with the characteristics of a classical Ca2+ sensor and, in contrast to the typical switch-like role controlled by protein exposure of the hydrophobic patches, indicate a different role of Ca2+ for target interactions of CML14. Instead CML36 showed the characteristics of a typical Ca2+ sensor. In conclusion our work provides new and interesting insights into the biochemical properties of Arabidopsis CML14 and CML36 that may be useful to future studies aimed at elucidating their precise physiological role.