Research

Our major scientific interest is centered on the understanding of protein function at the molecular level. Our work lies on the interface between biology, chemistry and physics: protein crystallography in combination with biochemistry and molecular biology constitute the core of our experimental methods. Two major lines of research are being pursued: I. structural biology and mechanistic studies of glycosyltransferases; and, II. signal transduction; special interest is given to understanding the molecular mechanisms underlying information transmission in proteins, using both prokaryotic and eukaryotic models. These include regulatory proteins involved in bacterial fatty acid synthesis, as well as trypanosomal Ser/Thr protein kinases involved in differentiation/virulence.

Structural glycobiology

We have made progress most notably in the structural studies of sialidases and trans-sialidases (TS) from trypanosomal parasites. Five articles have been published (Neres et al., 2007; Buchini et al., 2008; Damager et al., 2008; Buschiazzo & Alzari, 2008; Neres et al., 2009). I have also participated in the crystallographic aspects in collaboration with Dr Mary Jackson (Unité de Génétique Mycobactérienne - IP), solving the structure of the mannosyl-transferase PimA from Mycobacterium tuberculosis, essential for this pathogenic bacterium (Guerin et al., 2007).

Using our structural information on parasite sialidases, several collaborations have been recently concentrating in the rational design of T. cruzi TS inhibitors that could eventually evolve towards therapeutic applications, badly needed to combat Chagas’ disease. In this line, collaborative research is being pursued with Dr O Campetella (Universidad de San Martin, Argentina), concerning the use of neutralizing antibodies able to inhibit the trans-sialidase activity from T. cruzi. The crystal structure of TcTS in complex with a high-affinity monoclonal antibody, has been recently solved in our lab (manuscript in preparation).

Signal transduction

To understand the molecular means by which bacteria transduce signals, adapting to a changing environment, we are focused in elucidating the molecular mechanisms of certain key proteins involved in the regulation of lipid synthesis in Gram+ bacteria, using Bacillus subtilis as a model. This line of research has resulted in two high impact articles, one in the period being reported (Albanesi et al., 2009), which includes most of the work performed at the IP Montevideo. A third article is currently under revision in the Journal of Biological Chemistry (Trajtenberg et al., 2010). Several major discoveries have been made, predicting a fruitful development to be continued in the next few years. This project is being supported by the French Research Agency (ANR) and the Uruguayan National Research Agency (ANII). It has enabled us to strengthen a collaborative network with the teams of P Alzari and Michael Nilges (Unités de Biochimie Structurale and Bioinformatique Structurale, Institut Pasteur, Paris) and Prof. D de Mendoza (Dept of Microbiology, Instituto de Biologia Molecular y Celular de Rosario IBR, Rosario, Argentina). The main subject of our contribution concerns the structural study of DesK, a trans-membrane histidine kinase that, together with its cognate response regulator DesR, constitutes a two-component system, ultimately regulating the membrane’s fluidity in response to cold shock in B. subtilis and related species. The 3D structure of the entire cytoplasmic region of DesK from B. subtilis (Albanesi et al., 2009), determined in different functional configurations along the regulation cycle, allowed us to propose a mechanistic model that appears to be general for histidine kinase-mediated signal transduction. More recently, we have also been able to solve a high resolution structure of the ATP-binding domain of DesK (PDB 3EHG, Trajtenberg et al., submitted 2010). Comparative structural analyses allowed us to identify conserved regions suspected to be relevant in the autophosphorylation mechanism. Subsequent structure-based mutagenesis lead to the kinetic trapping of a putative transient state of autophosphorylating DesK, confirming the structural hypotheses.

We have recently applied for funding (Open call ACIP “Actions Concertées Inter-Pasteuriennes”, Institut Pasteur 2010 – under revision) with the project “Signal transduction in Leptospira virulence regulation: a multidisciplinary approach”. This project is going to be based at Montevideo, in collaboration with Dr. Albert Ko (Lab. Patologia e Biologia Molecular, Centro de Pesquisas Gonçalo Moniz, Fiocruz, Salvador – Brasil) and Dr. Mathieu Picardeau (Unité de Biologie des Spirochetes, Institut Pasteur, Paris).

Finally, we have made substantial progress in the project “Targeting the Leishmania kinome for the development of novel anti-parasitic strategies”, a multicentric collaborative initiative funded by the EU (FP7). We have solved a first serine/threonine protein kinase target from Leishmania major at 2.2 Å resolution, structural analyses are underway.