RICERCA ITALIANA     

Research program

Supramolecular complexes of sorcin in the generation and regulation of Calcium-dependent cellular functions

University Co-ordinator

Consiglio Nazionale delle Ricerche - ()

Research Unit Leader

Gianni Colotti

Description

The aim of the project is to investigate the molecular basis of the interactions between sorcin and some of its cardiac protein targets in order to unveil their physiological effects in the framework of the excitation-contraction-relaxation processes in the heart.
The research focus will be on RyR2 and NCX among calcium channels; sorcin will be employed as well as the isolated C-terminal calcium binding domain (SCBD), and site-specific mutants of residues located mainly in the D helix, which plays a relevant role in the information transfer process, and in other regions of the molecule.
The research project proposed by Unit Colotti also intends to contribute to the characterization of the interaction between sorcin and casein kinase II (CK2), a kinase endowed with antiapoptotic role that favours cellular proliferation and survival and that, together with sorcin, is overexpressed in Multi Drug Resistant transformed cells.

1. Sorcin–RyR interaction
Ryanodine receptors (RyRs) are high molecular weight (?565 kDa/monomer) homotetrameric proteins and among the most important endo/sarcoplasmic reticulum Ca2+ channels. In mammals, three RyR isoforms are known: RyR1, expressed mostly in the skeletal muscle, RyR2, typical of the cardiac tissue, and RyR3 which is ubiquitous, but present at higher concentrations in the brain. RyRs are involved in calcium release from endo/sarcoplasmic reticulum acting in clusters and therefore generating Ca2+ sparks (Cheng et al., 1993).
Sorcin interaction with RyRs is calcium dependent. Complex formation occurs via the sorcin C-terminal domain (Zamparelli et al., 2000) and determines channel inactivation as indicated by experiments on permeabilized myocytes where Ca2+-spark frequency, amplitude and duration were all significantly reduced (Lokuta et al., 1997; Seidler et al., 2003; Bers et al., 2004). To date only the interaction of sorcin with RyR1 and RyR2 has been assessed. Due to the high sequence identity of the three RyR isoforms, it is conceivable that sorcin interacts also with RyR3.
Previous studies carried out by Unit Colotti assigned an important role in the sorcin-RyR2 interaction to the D helix. In particular, the C-terminal region next to EF3 and to W105 is most likely involved in the interaction. Thus, substitution of W105 affects markedly the in vitro capacity of sorcin to interact with RyR2, while mutation of the other tryptophan residue (W99), located at the other end of the D helix near EF2, has a small effect (Colotti et al., 2006).
The RyR2 regions involved in interactions with its cytoplasmic molecular targets are located in the “foot region” (amino acids 1-4511); 4 different constructs of about 750-800 residues each will be cloned, expressed and purified by Unit Sorrentino. To identify and clone the RyR2 regions interacting with sorcin, Unit Colotti will take advantage of the known RyR2 regulatory regions, namely those regions that include the recognition sites for two inhibitory targets proteins, calmodulin (amino acids 3583-3603) and FKBP12 (amino acids 1-1937, amino acids 3788-4967). The inhibitory effect of sorcin on RyR2 activity leads to the hypothesis that sorcin may bind to RyR2 using these very same recognition regions. These will be cloned, expressed and purified by Unit Sorrentino which will map the RyR2 regions involved in the interaction.
On the other hand, Unit Colotti:

1.1 will assess the ability of the above mentioned RyR2 regions to interact with sorcin, using Surface Plasmon Resonance (SPR). This technique allows a real-time ligand-analyte interaction analysis that provides both kinetic information about complex formation (association and dissociation rates) and equilibrium constants. The SPR technique takes advantage of optical biosensors. The ligand is immobilized on a modified dextran matrix coated onto a thin metallic film, while the analyte flows into the flow cell. The interaction between the immobilized ligand and the analyte results in a change in refractive index that determines a shift in the angle at which the incident light is absorbed due to the SPR phenomenon. The cloned RyR2 regions will be immobilized and the sorcin sample will be injected (or vice versa) in the presence of different calcium concentrations. This approach will permit the identification of the channel region that recognizes sorcin and the determination of the kinetic and thermodynamic parameters of ligand-analyte complex formation and its dependence on the availability of calcium.

1.2 will express and purify sorcin mutants of residues located in the EF hands and in the D-helix, and the natural F112L variant (the F112L mutation has been associated with a familial form of cardiac hypertrophy);
will clone, express and purify mutants of potential phosphorylation sites (Ser80, Thr155 and Ser178 will be substituted by Asp residues to mimic their phosphorylated adducts);
will clone, express and purify mutants of hydrophobic residues in the G-helix and in the GH loop (F134A, F173A and F186A sorcin mutants). The relevant residues, based on the model for sorcin activation (Ilari et al. 2002), could be exposed on the molecular surface upon calcium binding and participate in the interaction with protein targets.
All the above mentioned mutants will be used in SPR experiments to identify the regions of sorcin involved in the interaction with RyR2.

1.3 will utilize analytical ultracentrifugation and (if necessary) dynamic light scattering techniques, to assess the molecular mass and thereby calculate the stoichiometry of the complexes.

1.4 to establish the effect of sorcin and its mutants on the RyR2 functional properties, Unit Colotti will carry out direct calcium sparks measurements on rabbit cardiomyocytes, in the presence or absence of wt sorcin and its mutants. The calcium transients will be revealed in the presence fluorophore Fluo3. These experiments will be carried out in the framework of a well established collaboration with Prof. Godfrey L. Smith at the University of Glasgow (Colotti et al., 2006).

1.5 will perform crystallization trials of the RyR2 suppressor and core domains which display 23-25% identity and 43-53% similarity with the corresponding domains of the IP3 receptor whose X-ray crystal structure is known (Bosanac et al. 2002; Bosanac et al., 2005). Intriguingly, in IP3 receptors the core region is involved in the binding of IP3, a process which is regulated by the suppressor domain. Crystallization trials will be performed also on sorcin-RyR fragments complexes. Unit Colotti will use synchrotron radiation to try and solve the three-dimensional structures of any X-ray quality crystals obtained.

1.6 will study the role played by phosphorylation in the regulation of sorcin and RyR, since it is known that both proteins are phosphorylated “in vivo”. To this end, sorcin mutants mimicking phosphorylated adducts (Asp residues in place of the canonical serine and threonine residues at the potential phosphorylation sites) will be designed and produced. Their interaction with RyR2 fragments will be characterized using the above mentioned techniques.

1.7 will extend the study of the topology of interaction to RyR1 and RyR3, that share a high sequence identity (about 70%) with RyR2. The sorcin-RyR1 interaction is known to occur (Zamparelli et al., 2000). Preliminary data obtained in collaboration with Unit Sorrentino show that sorcin interacts with RyR3 and regulates its activity.


2. Sorcin-NCX interaction

The Na+-Ca2+ exchanger (NCX), a transporter located in the cardiac plasma membrane which exchanges three Na+ for each Ca2+, interacts with sorcin and is activated upon complex formation (Seidler et al., 2003). In different phases of the excitation-contraction cycle, NCX can generate either depolarizing or repolarizing currents. However, under physiological conditions, it works mainly in the Ca2+ extrusion mode, driven mostly by calcium release from the SR (Bers et al., 2002).
NCX is a monomeric protein of 970 residues; according to the most recent structural model, it is organized in 9 transmembrane helices and a long cytoplasmic loop (about 520 amino acids) located between helices 5 and 6 (Hilge et al, 2006). The transmembrane region binds and exchanges Na+ and Ca2+, while the cytoplasmic loop has a regulatory role. The loop is formed by the XIP region (eXchanger Inhibitory Peptide) involved in Na+-dependent NCX inactivation, the calcium-binding CBD1 and CBD2 domains and the XIP binding site.
The Na+-Ca2+ exchanger is regulated by calcium, ATP, protein kinase A and protein kinase C (Shigekawa et al., 2001; Hilge et al., 2006). No information on the sorcin binding site is available to date.
Unit Colotti will investigate the following topics:

2.1 Immunoprecipitation experiments and experiments on isolated cardiomyocytes will be carried out on wt sorcin and on sorcin mutants to identify the region involved in the interaction with NCX. Voltage ramp measurements allowed demonstration of an increase in NCX activity following transfection with a sorcin-expressing vector (Seidler et al., 2003). Using this technique, in collaboration with Prof. G.L. Smith (University of Glasgow), the effect of wt sorcin, of the calcium binding domain (SCBD) and of sorcin mutants on NCX activity will be tested to define the region(s) involved in the interaction with NCX. In case the interaction takes place through the sorcin N-terminus, the residues involved in complex formation will be identified by affinity chromatography experiments. These will involve immobilization of synthetic peptides corresponding to different regions of the domain on a inert matrix; the immobilized peptides will be tested for their ability to bind NCX. In case the interaction takes place through the sorcin C-terminal domain, the topology of interaction will be analysed with a different approach, based on the functional characterization of site-specific mutants in the domain, e.g. mutants of the EF hands and of the D helix.

2.2 The regulatory loop region of NCX will be cloned, expressed, purified and used in immunoelectrophoresis and SPR experiments to investigate the dependence on calcium concentration and the thermodynamic and kinetic features of the sorcin-NCX complex formation. Subsequently, cloning, expression and purification of specific regions of the NCX loop will be carried out. These regions, in particular CBD1 and CBD2, will be tested for their ability to interact with sorcin by affinity chromatography and SPR.


3. Sorcin-CK2 interaction

Unit Colotti will collaborate with Unit Ruzzene to investigate the importance of sorcin in the development of Multi Drug Resistance (MDR). Unit Colotti will contribute to characterize the interaction between sorcin and casein kinase II (CK2), a ubiquitous kinase with an anti-apoptotic role, which favours proliferation and cell survival. Both the CK2 alpha-subunit and sorcin are overexpressed in transformed cells with MDR phenotype.
It is conceivable that both proteins (or possibly their complex) have a role in the establishment of this phenotype in view of recent studies by Unit Ruzzene which demonstrate the interaction in vitro between sorcin and the CK2 β subunit.
Unit Colotti will carry out the following studies in collaboration with Unit Ruzzene:

3.1 The role of sorcin phosphorylation by CK2 will be investigated by cloning, expressing and purifying sorcin mutants where Ser80 and Thr155, the potential phosphorylation sites, are altered by substitution with aspartic acid residues. The extent of sorcin phosphorylation will be measured by Unit Ruzzene in eukaryotic cell lines: cloning of sorcin in eukaryotic expression vectors will be carried out by Unit Colotti.

3.2 The characterization of the sorcin-CK2 interaction by means of a multichannel SPR instrumentation. The multichannel feature permits a fast set up, the coverage of a wide set of experimental conditions and the averaging of a large number of sensorgrams which leads to a better signal to noise ratio. These data will complement those obtained by Unit Ruzzene.

3.3 The effects of the interaction with CK2 on the functional properties of sorcin, such as calcium affinity and the ability to interact with other target proteins, will be assessed.