15 research outputs found
Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin
Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA
Computational studies towards the identification of novel rhodopsin-binding compounds as chemical chaperones for misfolded opsins
Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber’s congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber’s congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases
Production and characterization of SulP anion transporters
The main subject of this thesis is the Sulphate Permease (SulP) protein family that includes more than two hundred members, identified in archaea, bacteria, fungi, plants and animals. Many of these proteins have been functionally characterized: most are anion exchangers or transporters with different substrate specificities and distinct mechanism of action (Saier et al., 1999). In mammals, the SulP family, known as Solute Linked Carrier 26 (SLC26), is composed of eleven members with important roles in normal physiology (Mount and Romero, 2004).
The SulP proteins show a similar structural organization: a hydrophobic central core, which includes ten or twelve membrane helixes, and a less conserved C-terminal cytoplasmic portion that includes a STAS domain (Sulphate Transporter and Anti-Sigma factor antagonist domain). Despite the functional role of the STAS domain is still unclear, it appears to be crucial for the regulation of the transport activity (Ko et al., 2004; Zheng et al., 2005; Shibagaki and Grossman, 2006). Its fundamental role is also underlined by the fact that mutations that alter this domain in the SLC26 family can cause loss of function, resulting in serious genetic diseases, like diastrophic dysplasia or Pendred syndrome (Dawson and Markovich, 2005). No three-dimensional structure of STAS domains or full-length sequences is available for any SulP anion transporter.
One part of the work was focused on the production of different forms of the STAS domain from different species, for the biophysical and structural characterization. Another part of the SulP project was performed at the Johann Wolfgang Goethe University of Frankfurt (Germany) and aimed at the production of some full-length SulP proteins, by a cell-free expression system, an emerging technique for the large-scale production of membrane proteins.
In the last year, I was also involved in the crystallographic study of the Green Fluorescence Protein mutant, GFPmut2, in collaboration with Prof. Stefano Bettati of the University of Parma (Italy). The main aim of this work was the elucidation of the structural basis of the spectroscopic properties of this mutant, in particular with respect to changes in pH. The GFP chromophore can, in fact, exist either in a protonated or deprotonated state, with distinct spectral properties (Tsien, 1998). In a previous spectroscopic characterization, GFPmut2 (Ser65Ala, Val68Leu, Ser72Ala) was found more sensitive than the wild type GFP to pH changes in the physiological range (Chirico et al., 2002). The structures of GFPmut2 at pH 6 and pH 9 were determined at around 1.6 Å resolution, allowing the correlation between the spectral and structural properties.L’oggetto principale di questo lavoro di tesi è la famiglia dei trasportatori anionici SulP (Sulphate Permease), che comprende più di duecento membri identificati in archea, batteri, funghi, piante e animali. Molte proteine di questa famiglia sono state funzionalmente caratterizzate e agiscono da trasportatori o scambiatori di anioni, e differiscono per l’affinità verso il substrato e il meccanismo di trasporto (Saier et al., 1999). Nei mammiferi la famiglia SulP, conosciuta come "Solute Linked Carrier 26" (SLC26), è composta di undici membri che svolgono un ruolo fondamentale in molti processi fisiologici nell’uomo (Mount e Romero, 2004).
Tutte le proteine SulP possiedono un’organizzazione strutturale simile: una parte centrale idrofobica, che comprende dieci o dodici eliche di membrana e una porzione C-terminale citoplasmatica meno conservata, che include il dominio STAS (Sulphate Transporter and Anti-Sigma factor antagonist). Sebbene non sia ancora chiaro il ruolo funzionale di questo dominio nei trasportatori di anioni, esso sembra essere di cruciale importanza per la regolazione dell’attività di trasporto (Ko et al., 2004; Zheng et al., 2005; Shibagaki e Grossman, 2006). Il suo ruolo fondamentale è rilevato anche dal fatto che mutazioni che alterano questo dominio nei membri della famiglia SLC26 possono comprometterne seriamente la funzionalità , causando malattie genetiche gravi, come la displasia diastrofica o la sindrome di Pendred (Dawson and Markovich, 2005). Non sono ancora note strutture tridimensionali di nessun dominio o intera proteina SulP.
Una parte del lavoro è stata focalizzata sulla produzione di diverse varianti del dominio STAS da specie diverse, finalizzata alla caratterizzazione biofisica e strutturale. Una seconda parte del progetto, svolta presso la "Johann Wolfgang Goethe University" di Francoforte (Germania), ha riguardato la produzione di intere proteine SulP mediante la sintesi in vitro, una tecnica molto promettente per la produzione su larga scala di proteine di membrana.
Durante l’ultimo anno, mi sono anche dedicata allo studio cristallografico di un mutante della Green Fluorescent Protein, GFPmut2, in collaborazione con il gruppo del Prof. Stefano Bettati dell’Università di Parma. L’obiettivo principale di questo lavoro è stato definire le basi strutturali delle proprietà spettroscopiche di questo mutante, in particolare al variare del pH. Il cromoforo della GFP può, infatti, esistere sia in forma protonata che deprotonata (Tsien, 1998). Le proprietà spettroscopiche della GFPmut2 (Ser65Ala, Val68Leu, Ser72Ala) sono state in precedenza caratterizzate e, rispetto alla proteina wild type, sembra essere più sensibile alle variazioni di pH nell’intervallo fisiologico (Chirico et al., 2002). A tal fine, è stata determinata la struttura della GFPmut2, sia a pH 6 che a pH 9, con una risoluzione di circa 1.6 Å. Il confronto delle due strutture ha consentito la correlazione delle proprietà strutturali con quelle spettroscopiche
Basi strutturali delle proprietĂ di legame del dominio Stas di prestina
Prestina è una proteina di membrana della famiglia degli SLC26, responsabile dell'amplificazione del segnale acustico dei mammiferi, grazie alle sue proprietà di elettromotilità . Questo lavoro di tesi mira a comprendere le basi strutturali delle proprietà di legame e traslocazione degli anioni dalla posizione C-terminale di prestina (dominio STAS
The STAS domain of mammalian SLC26A5 prestin harbours an anion-binding site
none5nononeLolli, Graziano; Pasqualetto, Elisa; Costanzi, Elisa; Bonetto, Greta; Battistutta, RobertoLolli, Graziano; Pasqualetto, Elisa; Costanzi, Elisa; Bonetto, Greta; Battistutta, Robert
Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin
The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505-744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529-744], PreCD(L), and [529-720], PreCD(S)). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCD(L) is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin
Structure and single crystal spectroscopy of Green Fluorescent Proteins
Usually, spectroscopic data on proteins in solution are interpreted at molecular level on the basis of the three-dimensional structures determined in the crystalline state. While it is widely recognized that the protein crystal structures are reliable models for the solution 3D structures, nevertheless it is also clear that sometimes the crystallization process can introduce some "artifacts" that can make difficult or even flaw the attempt to correlate the properties in solution with those in the crystalline state. In general, therefore, it would be desirable to identify some sort of control. In the case of the spectroscopic properties of proteins, the most straightforward check is to acquire data not only in solution but also on the crystals. In this regard, the Green Fluorescent Protein (GFP) is an interesting case in that a massive quantity of data correlating the spectroscopic properties in solution with the structural information in the crystalline state is available in literature. Despite that, a relatively limited amount of spectroscopic studies on single crystals of GFP or related FPs have been described. Here we review and discuss the main spectroscopic (in solution) and structural (in crystals) studies performed on the GFP and related fluorescent proteins, together with the spectroscopic analyses on various FPs members in the crystalline state. One main conclusion is that "in cristallo" spectroscopic studies are useful in providing new opportunities for gathering information not available in solution and are highly recommended to reliably correlate solution properties with structural features. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State
Development of novel treatments against inherited blinding diseases Retinitis pigmentosa and Leber's congenital amaurosis [Abstract]
Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters
Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti- sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti- sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Ă… resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which-although previously considered merely as a generic linker between the domain and the last transmembrane helix-is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties