Research lines

Development of the SIRAH coarse-grained force field (Southamerican Initiative for a Rapid and Accurate Hamiltonian).
Our group develops and maintains one of the broadest coarse-grained force fields for existing biomolecular simulations. SIRAH (www.sirahff.com) uses a Top Down approach and a classic Hamiltonian, common to atomic force fields. SIRAH is freely distributed with easy-to-use analysis tools, parameters and topologies to simulate DNA, proteins, explicit solvent and phospholipids. Currently, representations are being developed for metal ions, glycans and RNA. This line is entirely developed by our group.

Development of FRET sensors for cyclic nucleotide and redox signalling pathways.
Using bioinformatics and structural modelling, together with coarse-grained simulations, we have developed a new generation of FRET sensors for signaling cAMP, cGMP and redox conditions. These biosensors allow us to reach an unprecedented spatial resolution since they can be genetically fused to the C-terminal of virtually any protein, directing them to any cell compartment. This research line continues with the design of new generations of biosensors in the framework of the ProTeMCA program and in collaboration with foreign experimental groups.

Flavivirus stability studies.
Using multiscale simulations, we study the different factors that affect the stability of viral particles (Virus-like Particles). The reduced computational cost of our simulation scheme allows us to perform comparative simulations of different flaviviruses varying the temperature and pH conditions. The availability of experimental structures of viral particles of Zika, Dengue, Japanese encephalitis (JEV) and tick-borne Encephalitis virus (TBEV) allows us to study the accessibility of different epitopes, helping to understand the mechanisms of viral neutralization by antibodies. Additionally, the computational approach enables the identification of amino acids involved in the acid firing mechanism of flaviviruses, which could contribute significantly to the development of vaccines through the creation of attenuated viruses. These studies are part of collaborations with national and foreign experimental groups.

Research lines

DBC1 as modulator of metabolic function.
The main focus of our research has been to continue to understand the role of the protein Deleted in Breast Cancer-1 (DBC1), a SIRT1 regulator, in the control of metabolism and metabolic diseases. In order to achieve that, we took four different scientific approaches.

A) We continued using the genetic deletion of DBC1 mouse model
as an experimental paradigm in metabolism regulation;
B) we engaged in studying how DBC1 function is regulated in vivo. C) We decided to generate a loxp/loxp DBC1 mouse model as a tool for tissue-specific knockout of DBC1. D) Based on our previous data that DBC1 regulates the “healthy obesity” phenotype, we began to search for secreted targets of DBC1 that may account for its effects.

Novel regulators of metabolism and metabolic diseases
: focus on Inflammation. Chronic inflammation has emerged in the past few years as a major player in the development of metabolic diseases, with accumulating evidences showing that both innate and adaptive immune cells are involved in the onset and progression of obesity, type II diabetes and atherosclerosis. During this period, we begun to work with two different proteins in context of chronic inflammation and metabolic diseases: SIRT6 and “TMEM176B. Research and development in anti-obesity drugs.

The development of novel compounds for pharmacological treatment
of metabolic diseases was a seminal part of our G5 proposal. We associated in close collaboration with Dr. Carlos Batthyany, who together with Dr. Virginia Lopez had already designed several compounds aimed to treat atherosclerosis. Together, we showed that these compounds are effective in vivo for atherosclerosis. In the course of this scientific collaboration, we designed together a novel family of compounds, one of which is showing striking results on prevention of obesity, insulin resistance and non-alcoholic liver steatohepatitis (NASH).

Research lines

Signaling and regulation in microorganisms
We are particularly interested in two component systems (TCS) in bacteria. TCSs are central in mediating signaling and regulation, almost ubiquitous in prokaryotes and archaea, they are also present in fungi and plants. Other regulatory proteins, such as one component systems, are also being studied. The general question behind this line of research is: how do cells use these sensory and regulatory proteins to detect extra- and intra-cellular signals, and then regulate specific functions? To answer this question, saprophytic bacteria (such as Bacillus subtilis and Leptospira biflexa) are studied, as well as pathogenic ones (Leptospira interrogans, L. borpetersenii, L. noguchii, Enterococcus faecium, Mycobacterium tuberculosis). In these models, several key biological processes are studied, such as the regulation of lipid synthesis (Albanesi et al., Proc Natl Acad Sci USA 2009, 106: 16185, Trajtenberg et al., J Biol Chem 2010, 285: 24892, Trajtenberg et al. al., mBio 2014, 5: e02105; Trajtenberg et al., eLife 2016, 5: e21422; Imelio et al., Bio-protocol 2017, 7: e2510; Lara et al., 2018 submitted), heme metabolism (Morero et al., Mol Microbiol 2014 , 94: 340), or virulence in pathogenicity (Adhikarla et al., Front Cell Infect Microbiol 2018, 8:45).

Molecular and structural biology of Leptospira
Different species of Leptospira cause leptospirosis. This zoonosis is the most widespread one around the world, reemerging as an important problem in human and animal health. In Uruguay, it is a significant issue, provoking cattle abortions and reproductive failure, and transmitting to humans causing acute disease. Our lab wants to elucidate the molecular mechanisms that determine and/or regulate virulence and pathogenicity in Leptospira. There are currently two main projects to do this: the study of the motility apparatus and systematic efforts to isolate and type local Leptospira strains in Uruguay.

i- The motility apparatus of Leptospira. Active translational motility of leptospira (swimming) is central for the virulence of the pathogenic species that cause leptospirosis. The essential organelle used for swimming is the flagellum. Our lab is currently studying the detailed molecular architecture of the flagellar appendage. The filament of these spirochetes’ flagella is confined within the periplasm, a unique feature shared by all Spirochetes, including bacteria like Treponema pallidum (the agent of syphilis) or Borrelia burgdorferi (Lyme disease). In collaboration with the Ko and Sindelar labs in Yale University, as well as the Picardeau lab at the Institut Pasteur (Paris), we have shown that the flagellar filament from Leptospira is much more complex compared to better-known bacterial models used so far as a paradigm of swimming motility in bacteria. As an example, filaments from Salmonella and other Enterobacteria are built as homopolymers of a single protein species (flagellin), whereas Leptospira comprise at least eight different proteins polymerized into the periplasmic filament assembly (Wunder et al., Mol Microbiol 2016, 101: 457; San Martin et al., Acta Crystallogr F 2017, 73:123; Wunder et al., Front Cell Infect Microbiol 2018, 8:130). We have recently crystallized two new proteins from both pathogenic and saprophytic Leptospira spp. (San Martín et al., Acta Crystallogr F 2017, 73:123),and solved their 3D structures (unpublished results), revealing novel protein folds. We are now moving forward towards their physiological interactions and interaction partners, enabling for the flagellar filament role in spirochete motility.

ii-Isolation and typing of autochthonous Leptospira spp. strains. This line is being developed in the context of a multicentric collaborative project, aims to isolating native strains from Leptospira spp. from biological samples obtained from infected cattle and other animal reservoirs. These isolates are typed by complementary techniques: classic serologic methods and novel molecular approaches, ultimately achieving greater sensitivity and specificity (Zarantonelli et al., PLoS Negl Trop Dis 2018, 12:e0006694). This effort has led to the creation of a biobank of strains of Leptospira, which up to now was not available in Uruguay, reporting the identity of the serovars that circulate in natural infections. This biobank will be useful in the formulation of more efficacious vaccines, in the improvement of diagnostic methods, as well as in further investigations of leptospirosis in Uruguay.

Collaborations
Our lab works in collaboration with Dr. Hugo Gramajo (Institute of Molecular and Cellular Biology, IBR, Rosario, Argentina) and his team, to elucidate crystallographic structures and mechanisms of action of Mycobacterium tuberculosis transcription factors (one component regulator systems), playing essential roles in regulating fatty acid metabolism in this pathogen.
We also collaborate with Dr. Mathieu Picardeau (Institut Pasteur, Paris, France) and Dr. Albert Ko (Yale University, New Haven, USA) on motility of Leptospira and pathogenesis mechanisms of these bacteria at the molecular level.
In addition, our lab integrates a multicentric consortium in Uruguay, working together with teams from the Veterinary Laboratories Division (“Miguel C Rubino” DILAVE) of the Ministry of Livestock, Agriculture and Fishery (Alejandra Suanes, Rodolfo Rivero), National Institute of Agricultural Research (Franklin Riet), Hygiene Institute at the Medical School (Felipe Schelotto), addressing problems of isolation, typing, diagnostics and genomics of Leptospira.

Research lines

Characterization of the origins of the immunological microenvironment in tumour progression and its correlation with the constitutive expression of AID in progressive patients with CLL. The objective is to corroborate if the constitutive expression of the enzyme cytidine deaminase (AID) during the evolution of the disease is a key event in the progression of CLL.

Deciphering of the effect of new inhibitors of kinases and proapoptotic molecules on the proliferative subpopulations of CLL. It aims to characterize the molecular and phenotypic effects of new kinase inhibitors and the action of proapoptotic molecules in proliferating subpopulations of the tumour clone during treatment.

The S100A9 protein as a new therapeutic target in CLL. Linkage of inflammation, microenvironment and clinical evolution. We aim to deepen the knowledge of the role of inflammation in the clinical evolution of patients with CLL.

Development of an artificial binding protein (ABP) platform to generate new prognostic and treatment tools in the LLC. The objective is to create a therapeutic tool (ABP) capable of simultaneously recognizing the tumor cell and recruiting natural killer cells (NK) to destroy the tumor clone.

Research lines

Study of the intracellular host-pathogen interaction with a systemic approach.
At the lab we have studied virulence factors of intracellular pathogens, as well as the host genes and routes necessary for the establishment of the infection, and the interface between them. We set out as main objectives the design of new strategies for the treatment or prevention of various infectious pathologies, such as Chagas disease, African trypanosomiasis, leishmaniasis, and tuberculosis

Determination of virulence factors necessary for infection with T.cruzi.
Regarding the establishment and persistence of T. cruzi infection, we have described a set of proteins related to the redox metabolism of trypanosomatids (triparedoxin cytosolic and mitochondrial peroxidase, triparredoxins, glutarredoxins, pteridin reductase), and we have characterized their structure and function. We are developing strategies for its inhibition, as well as its application in the development of therapeutic and preventive strategies.

Characterization of the changes in gene expression produced by intracellular parasites in human cells.
We have shown that there is a cellular reprogramming led by T. cruzi that allows the establishment and persistence of the infection in human cells. One of the main objectives we are currently pursuing is to identify host proteins, which, when inhibited, prevent T. cruzi infection. We are also extending this strategy for genomic and molecular biology studies in leishmaniasis, African trypanosomiasis, neosporosis and tuberculosis.

Research lines

Biophysical and structural characterization of the BLV capsid protein (BLV-CA).
The mechanism of BLV capsid formation through the self-assembly of thousands of copies of BLV-CA represents a key event in the retrovirus cycle. To understand this mechanism we have characterized the biochemical and biophysical properties that affect this process, and in collaboration with the Laboratory of Molecular and Structural Microbiology of the IPMontevideo we elucidate the three-dimensional structure of BLV-CA, showing that it is organized in a pseudohexagonal with an important conformational plasticity. On the other hand, we generated nano-antibodies directed against the viral capsid and we studied the effect of the interaction with capsid on the modulation of self-assembly.

Characterization of the interactions between CA-VLB and intracellular components of the host cell.
The transit of the viral capsid between the plasma membrane and the cell nucleus depends on the interaction with several cellular proteins. Other retroviral models have described restriction factors that disturb the conformation of the capsid generating anti-viral conditions. For the case of delta-retroviruses such as BLV we have no confirmed evidence on these mechanisms. In the HIV model it has been demonstrated that the interaction between capsid and nucleoporins would participate in this transit and in the entrance to the nucleus through the nuclear pore. Based on the ability of BLV-CA to self-assemble in vitro in tubular or supramacromolecular structures, and using affinity and mass spectrometry techniques, our objective is to identify and characterize the interactions between BLV-CA and the factors of the cellular guest involved in this traffic. We will also look for partners of BLV-CA that can act as innate immune sensors when analyzing cell lysates of permissive and non-permissive cells to BLV infection. The engineering cells generated by Francesca Di Nunzio in IP Paris will be used to identify new restriction factors or functional viral partners by mass spectrometry. We will also design and purify nanobodies against BLV-CA that will be labeled as microscopy approaches. The results obtained in BLV will then be transferred to the HTLV-1 research to define common and diverse mechanisms adopted by these delta retroviruses when establishing the viral infection.

Biochemical, structural and immunological characterization of the BLV envelope protein.
The BLV env complex plays a crucial role in determining viral infectivity, and is responsible for inducing fusion of viral and cell membranes after recognition of specific cell surface receptors.
We have optimized the expression of the soluble env ectodomain in Drosophila S2 cells with a natural and altered furin cleavage site. We induced protein expression and secretion in the supernatant by divalent metals, and we performed protein purification by affinity chromatography using a StrepTactin column followed by size exclusion chromatography. The quality control of the proteins was evaluated by mass spectrometry. This system should allow the production of sufficient material for crystallization assays, electron cryo-microscopy of isolated trimers and biophysical studies of the multimeric complex formed by the recombinant proteins.
Env is a major target of antiviral immune responses, generating both humoral neutralizing antibodies and T-cell specific adaptive immunity. It has been reported for other retroviruses that the presence of an immunosuppressive peptide (isu) in the Env glycoprotein structure could be important in their ability to immunomodulate immune responses. We are interested in studying the effect of amino acid modifications in the isu domain on humoral and cellular adaptation responses to the 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 hope to identify the modifications that reduce the immunosuppressive activity of this domain and, therefore, increase its immunogenicity. This result could be useful for the rational design of effective vaccines against this retrovirus.
Using purified BLV Env protein we have also developed a new ELISA test for use in the diagnosis of enzootic bovine leukosis. In collaboration with ATGen we are now generating a new EBL diagnostic kit that will be used in a field experiment with more than 50.000 dairy cows.

The identification of genetic traits is 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 using vaccines is promising, but there are still no effective products on the market. Considering that recent results show that BLV has a heritable component as high as 8%, a third strategy to control the disease would involve herd breeding by increasing the frequency of genotypes associated with resistance to infection.
We have analyzed in an experimental herd with high prevalence of BLV infection, a group of animals defined as “controllers” of the disease and characterized by low proviral load and low anti-BLV antibody titers. Other groups were defined as “non-controlling” with high proviral load and high specific antibody titers, and “negative” with no detectable presence of BLV.
Using peripheral blood mononuclear cells (PBMC) from these animals, we are characterizing, in collaboration with Natalia Rego and Hugo Naya of the IPMON Bioinformatics Unit, the transcriptomic representatives of these groups by massive mRNA sequencing (mRNAseq). We hope to identify genes and isoforms differentially expressed in control animals and interpret these differences in the context of biological processes, ontologies of sub or overrepresented metabolic pathways.

Research lines

Role of TORID-1 as an innate checkpoint in tumor immunity.

Role of TORID-1 and TORID-2 in the biology of chronic lymphocytic leukemia. (Collaboration with Pablo Oppezzo).

Characterization of small molecules capable of inhibiting or activating the conductance mediated by TORID-1 and 2.

Role of the intracellular protein TORID-1 in obesity and inflammation induced by obesity. (Collaboration with Carlos Escande).

Study of non-conventional anti-inflammatory drugs as immunomodulators in organ transplantation. (Collaboration with Carlos Batthyany).

Research lines

Small RNAs derived from tRNA and Y-RNAs as new molecular pathways in cancer and their potential as a source of new diagnostic biomarkers in cancer.
Our main line of research is oriented to the role of new classes of small regulatory RNAs derived from tRNA and Y-RNA as central actors in the proliferation and survival responses to stress, and its potential as new molecular mechanisms in the initiation and progression of cancer. The focus of our current research activities is on the extracellular biology of these regulatory RNAs, and their ability to work as signaling molecules between cells.
Part of our work has shown that these small RNAs are actively secreted by cells through extracellular vesicles or extra-vesicular fractions, being transferred to other cells, constituting a new mechanism of intercellular communication and transfer of genetic information. Additionally, its unique stability in the extracellular environment allows its detection in biological fluids, positioning itself as potential molecular biomarkers in human cancer.
Currently, in collaboration with the Clinical Oncology Service of the Hospital de Clínicas and the National Cancer Institute, we are studying the diagnostic value of small circulating RNAs derived from tRNAGlu, tRNAGly and Y4-RNA in patients with lung cancer.

Research and Development activities with health centers.
Our laboratory is currently participating in a series of initiatives to incorporate new diagnostic biomarkers in oncology, as well as genomic and molecular tools.
Genetic susceptibility to breast cancer. Together with the Hospital de Clínicas of the Faculty of Medicine (Udelar), we have incorporated in the oncology routine the study of mutations of a panel of 11 hereditary predisposition genes to breast and ovarian cancer. This procedure uses next-generation deep sequencing for analysis including the BRCA1 and BRCA2 genes among others.
Biomarkers of therapeutic prediction in lung cancer. Our laboratory performs fluorescent in situ hybridization (FISH) assays to detect translocations of the ALK gene, as a way to predict sensitivity to treatment with inhibitors (Crizotinib) in lung cancer.

Research lines

CCDC28B and BBS proteins in the regulation of ciliogenesis and cilia length.
In the lab we have been dissecting the biological role of a number of BBS proteins and the BBS-associated protein CCDC28B (coiled-coil domain containing protein 28B). In patients it was first reported that a reduction in CCDC28B protein levels, in the presence of mutations in BBS genes, results in a more severe presentation of the syndrome. CCDC28B interacts with a number of BBS proteins and we have demonstrated that it is a novel regulator of ciliary length, both in cells and in vivo in zebrafish. We know that the function of CCDC28B in the cilium relies, at least in part, on its interaction with SIN1 and the molecular motor kinesin 1. Currently, we continue characterizing CCDC28B with the goal of understanding the mechanim by which it regulates cilia.

Cilia targeting: similarities with nuclear transport.
Cilia are highly conserved organelles that protrude from the cellular plasma membrane while their interior is connected to the cytosol. However, the composition of cilia appears to be highly regulated and different from that of the membrane and cellular interior. Importantly, this particular composition is important for the function of the organelle. For example, receptors and mediators of different signaling cascades are concentrated inside the cilium and ciliary entry of molecules has been shown to be regulated. However, the mechanisms involved in directing molecules into the cilium as well as in mediating their entry are not completely understood. Different lines of evidence show a similarity with nuclear import. In this context, we have been studying the role of the nuclear import machinery in transporting proteins into the cilium, looking at proteins that are able to localize to both compartments. In particular, we have focused on Gli2, a transcription factor for Hedgehog signaling, that changes its localization in response to pathway activation. We were able to show that Gli2 used importins to enter both the nucleus and the cilium but importantly, it uses different importins in each case. We continue studying this process with the aim of gaining a better understanding of the mechanism.

Bardet-Biedl associated proteins in intracellular trafficking.
The functional characterization of BBS proteins has led us to uncover different extra-ciliary roles. For example, we showed that BBS7 is able to enter the nucleus where it modulates the activity of RNF2, a chromatin remodeling factor. Thus, defects in BBS7 result in changes in gene expression. More recently, we also documented a role for CCDC28B in the nucleus that is linked to cilia length regulation in a mechanism that we still do not completely understand. In collaboration with Dr. Norann Zaghloul at the University of Maryland, USA, we have shown that BBS proteins not only transport molecules to the base of cilia but have a broader role in intracellular transport and secretion of at least a subset of proteins. We have shown that BBS4 is required for the correct secretion of FSTL1, a function that we believe relevant to understand the etiology of BBS.

The role of BBS proteins and cilia in the development of ciliopathy associated phenotypes.
Dissecting the biological role of the BBS proteins and CCDC28B is critical to understand the mechanism underlying the development of BBS associated phenotypes. Therefore, in addition of studying the function of these proteins at a cellular and molecular level, we also evaluate their function in models that are relevant to the pathology. For example, we have studied the role of cilia in the development of the retina in zebrafish. Currently, we are studying the role of BBS proteins and cilia in adipogenesis and the development of obesity. Our results show that defects in BBS4 lead to a reduction in the secretion of FSTL1 (as previously mentioned). Importantly, we uncovered a novel role for FSTL1 in ciliogenesis and adipogenesis. This line of research is being followed in a collaboration with the laboratory of Dr. Escande in the context of the INDICyO Program.

Research lines

On biopharmaceuticals, the focus of study of the laboratory, we have developed specific kits and analytical methodologies base on requests from productive sector. Some of the projects that we are working on are:

Development of Methodologies to Quantify Proteins and DNA Contaminant Derivatives of the Host Cell in Recombinant Bio-Pharmaceuticals. Funded by the National Research and Innovation Agency. (Project ALIANZA – Laboratorio Celsius S.A. and IP Montevideo) (2011 – 2012).

Methodological development for quantification of immunogenicity generated by administration of Interferon beta1a in patients, by means of a Bioassay based on cell culture and Real Time PCR. Funded by Laboratorios Clausen S.A. (2010).

Participation in a Multicenter Study for the Determination of the Biological Potency of the First Filgrastim Standard of the United States Pharmacopeia (USP). (2012). (2012)