Protein structure, folding and function

Summary

We focus our interest on two important groups of proteins: molecular chaperones and flavoenzymes. We search for structure-function relationships as well as their metabolic insertion in the cell. These proteins are widely distributed among living organisms and they have relevant roles in cells in normal or stressful conditions.

We are particularly interested in the structural backgrounds that allow these proteins to fulfill their roles efficiently. We study the rules that govern substrate recognition, the activity regulation by the cell environment and the interaction with other cell proteins.

Our experimental strategies involve studies with wild type and mutant recombinant enzymes, natural and artificial substrates and isolated organelles. We use as models enzymes those from rice, pea, Arabidopsis thaliana and Leptospira interrogans as well as from other bacteria.

Research Lines

Flavoenzymes

Our model enzyme is the ferredoxin-NADP+ reductase (FNR). This enzyme catalyzes the electron transfer between mono- and bi-electronic obliged substrates in plastids, mitochondria and bacteria. We focus our interest on the catalytic mechanism of the enzyme at the molecular level. We search for 1) the structural elements involved in FAD binding and catalytic turnover, looking for the structural traits that allow for the large catalytic differences that distinguish plastidic from bacterial FNRs; 2) the catalytic mechanism of FNRs at the molecular level; 3) the functional insertion of a highly efficient FNR in Leptospira interrogans, Toxoplasma gondii and Plasmodium falciparum metabolisms, searching for their natural substrates.

Molecular chaperones

Protein import into chloroplasts and the maintenance of protein homeostasis in plastids are essential processes for the cell life during both normal and stressful conditions. Proteins destined to organelles are synthesized by cytosolic ribosomes as higher molecular mass precursors containing an N-terminal extension called transit peptide, which is subsequently removed by site-specific proteolysis after translocation. In addition, during both normal plant development and under stress conditions, significant protein turnover occurs largely through proteolysis. In all these mechanisms molecular chaperones are implicated. Among these, one of the most relevant families is the
Clp/Hsp100 chaperone family. We are interested in studying the interaction of molecular chaperones with artificial and natural substrates, with the aim of elucidating the molecular basis of substrate recognition as well as the regulation of these processes.

Selected Publications

  • Colombo, C. V., Ceccarelli, E. A. and Rosano, G. L. (2014). Characterization of the accessory protein ClpT1 from Arabidopsis thaliana: Oligomerization status and interaction with Hsp100 chaperones. BMC Plant Biology14 (1), 228
  • Sanchez-Azqueta, A., Catalano-Dupuy, D. L.; López-Rivero, A.; Tondo, M. L.; Orellano, E. G.; Ceccarelli, E. A. and Medina, M. (2014). Dynamics of the active site architecture in plant-type Ferredoxin-NADP+ reductases catalytic complexes. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1837(10): 1730-1738.
  • Soldano, A., Yao, H., Rivera, M., Ceccarelli, E. A. and Catalano-Dupuy, D. L. (2014). Heme-Iron Utilization by Leptospirainterrogans Requires a Heme Oxygenase and a Plastidic-Type Ferredoxin-NADP+ Biochimica et Biophysica Acta (BBA)-General Subjects 1840(11): 3208-3217.
  • Rosano GL and Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol.5:172. doi: 10.3389/fmicb.2014.00172.
  • Catalano-Dupuy DL, López-Rivero A, Soldano A, Ceccarelli EA. (2013) Redox proteins as targets for drugs development against pathogens. Curr Pharm Des., 19, 2594-605.
  • Sánchez-Azqueta A, Musumeci MA, Martínez-Júlvez M, Ceccarelli EA, Medina M. (2012) Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin-NADP(+) reductases. Biochim Biophys Acta. 1817, 1063-71.
  • Rosano G. L, Bruch E. M, Colombo, C. V. Ceccarelli E. A. (2012) Towards a unified model of the action of CLP/HSP100 chaperones in chloroplasts. Plant Signaling & Behavior, 7, 672-674.
  • Bruch EM, Rosano GL, Ceccarelli EA. (2012) Chloroplastic Hsp100 chaperones ClpC2 and ClpD interact in vitro with a transit peptide only when it is located at the N-terminus of a protein. BMC Plant Biol. doi: 10.1186/1471-2229-12-57.
  • Catalano-Dupuy D.L., Musumeci M.A., López-Rivero A., Ceccarelli E.A. (2011) A highly stable plastidic-type ferredoxin-NADP(H) reductase in the pathogenic bacterium Leptospira interrogans. PLoS One, 6: e26736. doi:10.1371/journal.pone.0026736.
  • Tondo M.L., Musumeci M.A., Delprato M.L., Ceccarelli E.A., Orellano E.G. (2011) Structural-functional characterization and physiological significance of ferredoxin-NADP+ reductase from Xanthomonas axonopodis pv. citri. PLoS One, 6(11):e27124.
  • Rosano G. L, Bruch E. M, Ceccarelli E. A. (2011) Insights into the CLP/HSP100 chaperone system from chloroplasts of Arabidopsis thaliana. J Biol Chem., 286, 29671-80.
  • Musumeci, M. A., Botti, H., Buschiazzo, A. and Ceccarelli, E. A. (2011) Swapping FAD Binding Motifs Between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases. Biochemistry, 50, 2111-2122
  • Carrillo, N., Ceccarelli E. A., Roveri O. A. (2010) Usefulness of Kinetic Enzyme Parameters in Biotechnological Practice. Biotechnology & Genetic Engineering Reviews. Biotechnology & Genetic Engineering Reviews, 27, 367-382.
  • Rosano, G. L., Ceccarelli, E. A. (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial Cell Factories, 8:41
  • Paladini, D., Musumeci, M. A., Carrillo, N., Ceccarelli, E. A. (2009) Induced-Fit And Equilibrium Dynamics for High Catalytic Efficiency in Ferredoxin-NADP(H) Reductases. Biochemistry, 48, 5760-5768.
  • Musumeci, M. A., Arakaki, A. K., Rial, D. V., Catalano-Dupuy, D. L., Ceccarelli, E. A. (2008). Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by the amino acid volume around the catalytic site. FEBS J., 275, 1350-1366.
  • Ceccarelli, E.A., Carrillo, N., Roveri, O. A. (2008) Efficiency function for comparing catalytic competence. Trends Biotechnol., 26, 117-118.
  • Nascimento, A. S., Catalano-Dupuy, D. L., Bernardes A., de Oliveira Neto., Santos, M.A., Ceccarelli, E. A., Polikarpov, I. (2007) Crystal Structures of Leptospira interrogans FAD-containing Ferredoxin-NADP+ Reductase and its complex with NADP+ . BMC. Struct. Biol., 7, 69.

Grants

  •  Proyecto de investigación conjunta “Mecanismos catalíticos en flavoenzimas: clave para su utilización biotecnológica o terapéutica”. Entidad financiadora: Ministerio de Ciencia e Innovación de España. Entidades participantes: Universidad de Zaragoza – IBR. Investigadora responsable: Prof. Dra. Milagros Medina, U. de Zaragoza, España.
  • PICT 2015-2955, ANPCYT, Mincyt, Argentina. “Proteostasis en cloroplastos de plantas. Regulación y especificidad del sistema Clp."
  • PIP CONICET 2012 Nro. 112-020120100345, del Consejo Nacional de Investigaciones Científicas y Técnicas, “Estructura y función de chaperones moleculares y flavoenzimas”.
  • PICT 2012 Nro. 1841, ANPCYT, Mincyt, Argentina. “Estudio de la ferredoxina-NADP reductasa altamente eficiente de Leptospira interrogans. Elucidación de su/s rol/es en el metabolismo del patógeno”·
  • Cooperación Internacional CONICET-NSF, con la Universidad de Kansas, Resolución 993/13 “The heme oxygenase of Leptospira interrogans: structural basis for its catalytic mechanism”.
  • Proyecto de investigación conjunta “Mecanismos catalíticos en flavoenzimas: clave para su utilización biotecnológica o terapéutica. BIO2010-14983”. Entidad financiadora: Ministerio de Ciencia e Innovación de España. Entidades participantes: Universidad de Zaragoza – IBR. Desde octubre, 2010 hasta octubre, 2013. Investigadora responsable: Prof. Dra. Milagros Medina, U. de Zaragoza, España.

Director

Array ( [4609] => Array ( [width] => 3378 [height] => 3240 [file] => 2013/12/Ceccarelli1.jpg [sizes] => Array ( [thumbnail] => Array ( [file] => Ceccarelli1-290x145.jpg [width] => 290 [height] => 145 [mime-type] => image/jpeg ) [medium] => Array ( [file] => Ceccarelli1-300x287.jpg [width] => 300 [height] => 287 [mime-type] => image/jpeg ) [large] => Array ( [file] => Ceccarelli1-1024x982.jpg [width] => 1024 [height] => 982 [mime-type] => image/jpeg ) ) [image_meta] => Array ( [aperture] => 2.8 [credit] => Lucia Ceccarelli [camera] => Canon EOS 60D [caption] => [created_timestamp] => 1380665829 [copyright] => [focal_length] => 50 [iso] => 500 [shutter_speed] => 0.0125 [title] => ) [ID] => 4609 [name] => Ceccarelli1.jpg [path] => /home/ibrconi/public_html/wp-content/uploads/2013/12/Ceccarelli1.jpg [url] => http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1-290x145.jpg [full_url] => http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1.jpg [title] => Ceccarelli [caption] => [description] => [alt] => [srcset] => http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1-300x287.jpg 300w, http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1-1024x982.jpg 1024w ) )
http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1.jpg
http://www.ibr-conicet.gov.ar/wp-content/uploads/2013/12/Ceccarelli1-150x150.jpg

Ceccarelli, Eduardo A.
Core CCT
Email: ceccarelli@ibr-conicet.gov.ar
Phone: +54 341 4237070
Office Extension: 640
Laboratory Extension: 612

Researchers

  • Daniela Catalano Dupuy
  • Germán Rosano

Postdoctoral fellows

  • Cecilia Balaban
  • Clara Victoria Colombo

Doctoral fellows

  • Paula Monchietti
  • Dianela Aguilar
  • Ivana Parcerisa
  • Alejo Cantoia

Undergraduate Students

  • Alejo Cantoia
  • Virginia Cointry

Imágenes

Estrategia para determinar la interacción de chaperones con péptidos. Las proteínas unidas son recuperadas mediante fijación a una resina que une GST (glutatión reductasa).

Modelo tripartito de unión entre la enzima FNR y sus dos sustratos: ferredoxina (Fd) y NADP(H).