Protein Biophysics Unit
- Otto Pritsch (PhD, Head)
- Gonzalo Obal (Technical Assistant, PhD student)
- Federico Carrión (Technical Assistant, MSc student)
- Sergio Bianchi (MD, MSc, PhD, posdoc)
- Lorena Tomé (MSc, PhD student)
- Natalia Olivero (PhD student)
- Andrés Addiego (MSc student)
- Thermodynamic analysis of protein-protein and protein-ligand interaction through determination by isothermal titration microcalorimetry (ITC) of binding constants (KB), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS).
- Thermodynamic analysis of conformational changes of proteins including assessment of stability and folding of recombinant proteins through differential scanning microcalorimetry (DSC) to study a wide range of thermal transitions in biological systems, to determine melting temperatures as well as thermodynamic parameters associated to these changes.
- Kinetic analysis of protein – ligand interaction through Surface Plasmon Resonance (SPR) measurements, determination of kinetic association (kass) and dissociation (kdiss) constants.
- Determination of the hydrodynamic radius of macromolecules or particles through dynamic light scattering measurements coupled to size exclusion chromatography SEC-HPLC.
During the period 2007-2014, we started at IPMONT a project focused on the study of viral pathogenesis of human and bovine chronic lymphocytic leukemias.
We have developed two main research axes:
First, we proposed to search for a candidate virus as aetiological agent of human Chronic Lymphocytic Leukaemia (CLL), by combining high-throughput sequencing and digital subtraction. With this aim we characterized at the molecular level a group of 80 CLL patients (Bianchi et al, 2010), and thereafter, B-cell transcriptomes from CLL patients and healthy donors were sequenced by using the 454 Life Sciences pyrosequencing technology and Illumina technology. Despite an in-depth analysis of the CLL transcriptome reaching more than 100 million sequences, we have not found evidence for a putative viral candidate in CLL (Rego et al, 2012).
Second, we started to study an animal model for virus-induced Chronic Lymphocytic Leukemia. Enzootic Bovine Leukemia (EBL) is an infectious disease caused by an oncogenic member of the genus Deltaretrovirus of the family Retroviridae, the Bovine Leukemia Virus (BLV), which affects >60% of dairy cattle in Uruguay. Most infections are subclinical, but 30% of infected cattle develop persistent lymphocytosis, and 5% develop lymphosarcomas. At the moment, no vaccine against BLV is available. In order to gain insight into the degree of genetic variability of BLV in our country we have performed a phylogenetic analysis of Env sequences and revealed the presence of seven BLV genotypes in the South American region (Moratorio et al, 2010). We also performed a detailed molecular analysis of complete bovine leukemia virus genomes isolated from B-cell lymphosarcoma, and compared with other BLV full-length sequences from other clinical manifestations (Moratorio et al, 2013). In parallel we developed a rapid and sensitive real time PCR assay using SYBR green chemistry to detect and quantify BLV proviral DNA from blood obtaining an increased sensitivity over the ELISA and AGID tests (Rama, 2010).
We also initiated the characterization of the main BLV proteins at the molecular and structural levels. In particular, we analyzed the self-assembly process of the purified recombinant BLV capsid (BLV-CA) protein providing the first description of their assembly properties. On the other hand, BLV-CA full-length and separate N- and C-terminal domains were expressed and purified to homogeneity. In order to obtain insights into the detailed molecular structure and self-assembly process of a native, non-engineered retroviral CA, we solved the crystal structure of the mature BLV-CA at 2.75Å resolution,showing a 2D hexagonal lattice displaying both lateral 3-fold and 2-fold interactions between asymmetric and plastic CA hexamers. This work was done in collaboration with IPMont Protein Cristallography Unit. (Obal et al, submitted).
In the context of this project we have organized a multidisciplinary group to work on BLV, funded by the Institut Pasteur de Montevideo, the National Institute of Agronomic Research of Uruguay (INIA), the Universidad de la República de Uruguay and the Centre National de la Recherche Scientifique (CNRS, France). Moreover, we have participated in the First Latin American Workshop on EBL (2012) and created the Regional Network of EBL that integrates a diverse group of research laboratories in Latin America. In parallel, the IPMONT is engaged as a founding member of the network “Center for Structural Biology in the Mercosur” – CeBEM (http://www.cebem.org.ar). Structural virology is one of the areas that need to be developed in our region, with anticipated impact in scientific and medical terms.
Principal and specific aims
In general, retroviruses use very similar principles in their biological cycles: assembly and budding of an immature particle, proteolytic capsid maturation, entry through membrane fusion via interactions of the envelope glycoprotein complex with a cellular receptor, reverse transcription of the viral genome, mature capsid uncoating, transport of the pre-integration complex into the nucleus and integration of the provirus. The principal aim of this proposal will focus on structural and functional studies on BLV, attempting to understand the structural bases of relevant functional phenomena of the viral biology.
Our specific scientific aims are:
1. To characterize the biochemical and structural bases of BLV envelope protein.
The BLV env complex plays a crucial role in determining viral infectivity, being responsible for inducing fusion of viral and cellular membranes after recognition of specfic cell-surface receptors.
To study this issue we will carry out the following specific tasks:
1.1. Cloning and expression of BLV-env glycoprotein in Drosophila S2 cells: We have optimized the expression of the soluble env ectodomain in Drosophila S2 cells, by cloning the codon-optimized env gene precursor in plasmid pT350. The env protein is truncated upstream the TM segment, and we have designed different constructs with a natural and an altered furin cleavage site. Protein expression and secretion into supernatant was induced by divalent metals, and protein purification was performed by affinity chromatography using a StrepTactin column followed by size exclusion chromatography. Protein quality control was assessed by mass spectrometry. This system should allow the production of sufficient material for crystallization trials, electron cryo-microscopy of isolated trimers, and biophysical studies of the multimeric complex formed by the recombinant proteins.
1.2. Analysis of the oligomeric structure of the expressed proteins: In order to determine the oligomeric state of the expressed BLV Env proteins, size exclusion chromatography, sedimentation analysis and light scattering we will be performed.
1.3. Crystallization trials of purified recombinant Env protein: data collection, structure determination and refinement: Crystallogenesis conditions will be screened with a robotic platform, and initial hints optimized as needed. Crystal quality will be checked with a home X ray source. Complete data sets will be collected at synchrotron facilities (Soleil, ESRF) and processed according to standard single crystal methods. Structures will be determined using Se-Met labeled proteins, and anomalous diffraction phasing techniques. Model refinement and validation will be carried out according to established methods.
1.4. Cryo-electron tomography of recombinant Env proteins. The IP has recently obtained funding through the “investissements d’avenir” (EquipEx) program to be equipped with a 300keV microscope for high resolution 3D studies on the different assemblies (capsid protein tubes, virus particles). Because the retroviral particles are pleomorphic, the studies will involve cryo-electron tomography with sub-tomogram averaging.
2. To characterize the biophysical and structural bases of BVL capsid self-assembly.
Like other retroviruses, assembly of BLV virions is driven by Gag, a polyprotein precursor composed of three major domains: MA (matrix), CA (capsid), and NC (nucleocapsid). After particle budding, the virus-encoded protease PR cleaves Gag and releases the individual domains: the N-terminally myristoylated MA remains anchored at the viral envelope, NC condenses with the viral RNA, and CA spontaneously self-assembles to form a closed structure: the mature “core”or capsid. This dramatic structural rearrangement, known as maturation, is essential for infectivity, and thus constitutes an attractive target for novel antiretroviral strategies. The mechanism of viral capsid formation via self-assembly of thousands of copies of the capsid protein (CABLV) represents a key event in the retrovirus cycle.
To study this issue we will carry out the following tasks:
2.1. To perform a comprehensive characterization of the biophysical properties of the CABLV assembly process: We will explore a wide range of conditions, to obtain a complete characterization of the parameters affecting the polymerization reaction. Particularly, we will focus on analyzing the effect of compounds in near-physiological conditions mimicking the virus intra-particle environment.
2.2. To elucidate the 3D structure of the CABLV protein. X-ray crystallography will be used to determine the atomic structure of the individual N-terminal and C-terminal domains, which will provide a high resolution view of the full-length mature protein. As mentioned above, we have already advanced in this direction, although further work is needed. Crystallogenesis conditions will be screened with a robotic platform. X ray diffraction data will be collected on our home source (microsource rotating anode generator) or synchrotron facilities (Soleil, ESRF) as needed. Structures will be determined using halogen (iodide, cesium) quick-soak derivatives, or Se-Met labeled proteins, and anomalous diffraction phasing techniques. Model refinement and validation will be carried out according to established methods. The high-resolution structures of full length and individual CABLV domains will provide a substrate for testing/redirecting hypotheses.
2.3. To characterize the ultra-structural morphology of in vitro assemblies of wild-type CABLV as well as engineered mutants. We will use cryo-electron microscopy and image reconstruction analysis of the in vitro assemblies to obtain electron density maps onto which mapping the crystallographic structures (to be done in collaboration with IPParis).
2.4. To perform structure/function analyses of the CABLV assembly process by mapping residues/surfaces functionally relevant for polymerization via extensive site-directed mutagenesis and in vitro effects on assembly. Structural information will guide the design of mutants and further engineering of CABLV. In solution functional studies and ultrastructural analysis using electron microscopy, will complement these studies on selected CABLV variants.
3. To characterize the immunomodulatory activity of BLV envelope glycoprotein.
Env is one of the main targets of the antiviral immune responses, generating both humoral neutralizing antibodies and T-cell specific adaptive immunity. It has been reported for other retrovirus that the presence of an immunosuppressive (isu) peptide in Env glycoprotein structure could be important in their ability to immunomodulate immune responses.
We propose to study the effect of amino acid modifications in the isu domain in humoral and cellular adaptive responses against challenge with modified Env glycoproteins. This will allow us to understand one of the mechanisms involved in the generation of resistance used by BLV to escape the antiviral immune response. On the other hand, we also expect to identify the modifications that reduce the immunosuppressive activity of this domain and therefore increase their immunogenicity. This result could be useful for the rational design of effective vaccines against this retrovirus.
To study this issue we will carry out the following tasks:
3.1. Expression and purification of the BLV wild-type and isu-mutant Env glycoprotein protein in Drosophila S2 cells.
3.2. Analysis of the in vitro and in vivo cytokine expression profiles in the presence of “isu mutant” versus “wild type” Env glycoprotein.
3.3 Characterize the antibody response in vivo after immunization with “isu mutant” versus “wild type” Env glycoprotein.
4. To identify the genetic characteristics associated with natural control of EBL.
Given the high prevalence of EBL in Uruguay, the strategy to eradicate the disease implemented in Europe and Oceania, is impracticable in our country. An alternative control strategy by using vaccines is promising, but there are still no effective products on the market. Taking into account that recent results show that EBL has a heritable component that reaches 8%, a third strategy to control disease would involve breeding herds by increasing the frequency of genotypes associated with resistance to infection.
4.1. Identify in herds with high prevalence of BLV infection, a group of animals defined as “controllers” of the disease and characterized by low proviral load and low titers of anti-BLV antibodies. Another group defined as “non-controllers” with high proviral load and high titers of specific antibodies.
4.2. Characterize the transcriptomes of peripheral blood mononuclear cells (PBMC) by massive sequencing of mRNA (RNAseq) obtained from controller and non-controller animals.
4.3. Identify genes and isoforms differentially expressed in “controller” as compared to “non-controllers” animals. Interpret these differences in the context of biological processes, metabolic pathways ontologies sub- or overrepresented.
Training of PhD degree students
- Gonzalo Obal, PhD Student, PEDECIBA. “Estudios Biofísicos y Estructurales del Ensamblado de la Cápside del Virus de la Leucemia Bovina” Director: O. Pritsch. Defense date Septembre 2014.
- Lorena Tomé, PhD Student, PEDECIBA. “Estudio de la interacción entre el Virus de la Leucosis Bovina y la célula hospedera”. Directors: J. Arbiza, O. Pritsch. Defense date Septembre 2014.
- Rodrigo Puentes, PhD Student, Faculty of Veterinary. “Epidemiología molecular y efecto de la presencia del virus de la Leucosis bovina enzoótica en animales asintomáticos sobre parámetros productivos, reproductivos y sanitarios”. Directors: Silvia Llambí, Gonzalo Moratorio and Otto Pritsch.
Training of Master degree students
- Federico Carrión, Master in Science, Faculty of Science. “Desarrollo de kits para genotípado de SNPs en los genes humanos, FII, FV y MTHFR , utilizando la tecnología PCR en Tiempo Real”. Directors: Andrés Abin and Otto Pritsch.
- Andrés Addiego, Master in Biomedicine, Faculty of Medicine. “Desarrollo y análisis comparativo de una nueva herramienta para el diagnóstico de la Leucosis Enzoótica Bovina; Impacto del descenso de anticuerpos anti-VLB circulantes en el periparto para el diagnóstico serológico” Directors: O. Pritsch. Defense date: March 2013.
- “International Associated Laboratory on Structural Virology”. Centre National de la Recherche Scientifique – IPMont-LIA. Period: January 2014 – December 20176. Felix Rey, CNRS URA 3015 Virology, Institut Pasteur, Paris – Otto Pritsch. Institut Pasteur de Montevideo. Amount granted: 15.000 euros / year.
- “Desarrollo de nuevos métodos para el diagnóstico del Virus de la Leucosis Bovina”. Ministerio de Industria y Energía – Dirección Nacional de Industria, uruguay. Period: December 2012 – December 2013. PI: Otto Pritsch. Amount granted 17.000 USD.
- Producción y Caracterización de Inmunógenos contra el Virus de la Leucosis Bovina. Proyecto CSIC I + D 2014, Universidad de la República. 2014 – 2017. PI Otto Pritsch. Amount granted 30.000 USD
- Identificación de marcadores moleculares asociados con la resistencia a la infección por el Virus de la Leucosis Bovina mediante análisis transcriptómico de individuos controladores de la carga viral. Fondo Sectorial Innovagro 2013, 2014 – 2016. PI Otto Pritsch. Amount granted 100.000 usd
- Doctoral Fellowship – Lorena Tomé – 2012 – 2014 – ANII.
- Tinoco LW, Fraga JL, Anobom CD, Zolessi FR, Obal G, Toledo A, Pritsch O, Arruti C. Structural characterization of a neuroblast-specific phosphorylated region of MARCKS (2014) Biochimica et Biophysica Acta – Proteins and Proteomics, 1844 (4), pp. 837-849. – IF: 1.094
- Morero NR, Botti H, Nitta KR, Carrión F, Obal G, Picardeau M, Buschiazzo A. HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes. Mol Microbiol. 2014 Oct;94(2):340-52. Epub 2014 Sep 15. PubMed PMID: 25145397. – IF: 5.026
- Correa A, Pacheco S, Mechaly AE, Obal G, Béhar G, Mouratou B, Oppezzo P, Alzari PM, Pecorari F. Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins). PLoS One. 2014 May 13;9(5) eCollection 2014. – IF: 3.534
- Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P. Generation of a vector suite for protein solubility screening. Front Microbiol. 2014 Feb 25. eCollection 2014. – IF: 3.941