Expression and variability of molecular chaperones in the sugarcane expressome

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. (c) 2006 Elsevier GmbH. All rights reserved

Identification and in silico expression pattern analysis of Eucalyptus expressed sequencing tags (ESTs) encoding molecular chaperones

Expressed Sequence Tags (ESTs) sequencing provides reliable and useful information concerning gene expression patterns in the genomic context. Our group used bioinformatics to identify and annotate 5'EST-contigs belonging to the molecular chaperones within the Eucalyptus Genome Sequencing Project Consortium (FORESTs) database. We found that 1,959 5'EST-contigs, or approximately 1.6% of the total 5'EST-contigs, encoded chaperones, emphasizing their biological importance. About 55% of the chaperones that we found were Hsp70 chaperones and its co-chaperones, 18% were Hsp90 chaperones, 15% were Hsp60 and its co-chaperone, 8% were Hsp100 chaperones, and 4% were Small Hsps. We also investigated the digital expression profile of the chaperone genes to gain information on gene expression levels in the different libraries and we found that molecular chaperones may have differential expression. The results discussed here give important hints about the role of chaperones in Eucalyptus cells

The C-terminal region of the human p23 chaperone modulates its structure and function

The p23 protein is a chaperone widely involved in protein homeostasis, well known as an Hsp90 co-chaperone since it also controls the Hsp90 chaperone cycle. Human p23 includes a β-sheet domain, responsible for interacting with Hsp90; and a charged C-terminal region whose function is not clear, but seems to be natively unfolded. p23 can undergo caspase-dependent proteolytic cleavage to form p19 (p231-142), which is involved in apoptosis, while p23 has anti-apoptotic activity. To better elucidate the function of the human p23 C-terminal region, we studied comparatively the full-length human p23 and three C-terminal truncation mutants: p231-117; p231-131 and p231-142. Our data indicate that p23 and p19 have distinct characteristics, whereas the other two truncations behave similarly, with some differences to p23 and p19. We found that part of the C-terminal region can fold in an α-helix conformation and slightly contributes to p23 thermal-stability, suggesting that the C-terminal interacts with the β-sheet domain. As a whole, our results suggest that the C-terminal region of p23 is critical for its structure-function relationship. A mechanism where the human p23 C-terminal region behaves as an activation/inhibition module for different p23 activities is proposed.The p23 protein is a chaperone widely involved in protein homeostasis, well known as an Hsp90 co-chaperone since it also controls the Hsp90 chaperone cycle. Human p23 includes a β-sheet domain, responsible for interacting with Hsp90; and a charged C-termi5655767FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2011/23110-0; 2012/50161-8SEM INFORMAÇÃOEchtenkamp, F.J., Freeman, B.C., (2014) The Molecular Chaperones Interaction Networks in Protein Folding and Degradation, pp. 207-232. , W.A. Houry, Springer New YorkJohnson, J.L., Toft, D.O., (1994) J. Biol. Chem., 269, pp. 24989-24993Johnson, J.L., Beito, T.G., Krco, C.J., Toft, D.O., (1994) Mol. Cell. Biol., 14, pp. 1956-1963Johnson, J.L., Toft, D.O., (1995) Mol. Endocrinol., 9, pp. 670-678Felts, S.J., Toft, D.O., (2003) Cell Stress Chaperon, 8, pp. 108-113Echtenkamp, F.J., Zelin, E., Oxelmark, E., Woo, J.I., Andrews, B.J., Garabedian, M., Freeman, B.C., (2011) Mol. Cell, 43, pp. 229-241Kobayashi, T., Nakatani, Y., Tanioka, T., Tsujimoto, M., Nakajo, S., Nakaya, K., Murakami, M., Kudo, I., (2004) Biochem. J., 381, pp. 59-69Tanioka, T., Nakatani, Y., Semmyo, N., Murakami, M., Kudo, I., (2000) J. Biol. Chem., 275, pp. 32775-32782Rao, R.V., Niazi, K., Mollahan, P., Mao, X., Crippen, D., Poksay, K.S., Chen, S., Bredesen, D.E., (2006) Cell Death Differ., 13, pp. 415-425Gausdal, G., Gjertsen, B.T., Fladmark, K.E., Demol, H., Vandekerckhove, J., Doskeland, S.O., (2004) Leukemia, 18, pp. 1989-1996Mollerup, J., Berchtold, M.W., (2005) FEBS Lett., 579, pp. 4187-4192Weaver, A.J., Sullivan, W.P., Felts, S.J., Owen, B.A.L., Toft, D.O., (2000) J. Biol. Chem., 275, pp. 23045-23052Weikl, T., Abelmann, K., Buchner, J., (1999) J. Mol. Biol., 293, pp. 685-691Forafonov, F., Toogun, O.A., Grad, I., Suslova, E., Freeman, B.C., Picard, D., (2008) Mol. Cell. Biol., 28, pp. 3446-3456Martinez-Yamout, M.A., Venkitakrishnan, R.P., Preece, N.E., Kroon, G., Wright, P.E., Dyson, H.J., (2006) J. Biol. Chem., 281, pp. 14457-14464Bose, S., Weikl, T., Bugl, H., Buchner, J., (1996) Science, 274, pp. 1715-1717Freeman, B.C., Toft, D.O., Morimoto, R.I., (1996) Science, 274, pp. 1718-1720Neckers, L., (2007) J. Biosci., 32, pp. 517-530Gava, L.M., Ramos, C.H.I., (2009) Curr. Chem. Biol., 3, pp. 10-21Da Silva, V.C.H., Ramos, C.H.I., (2012) J. Proteomics, 75, pp. 2790-2802Simpson, N.E., Lambert, W.M., Watkins, R., Giashuddin, S., Huang, S.J., Oxelmark, E., Arju, R., Garabedian, M.J., (2010) Cancer Res., 70, pp. 8446-8456Young, J.C., Hartl, F.U., (2000) EMBO J., 19, pp. 5930-5940Panaretou, B., Siligardi, G., Meyer, P., Maloney, A., Sullivan, J.K., Singh, S., Millson, S.H., Prodromou, C., (2002) Mol. Cell, 10, pp. 1307-1318Richter, K., Walter, S., Buchner, J., (2004) J. Mol. Biol., 342, pp. 1403-1413Ali, M.M.U., Roe, S.M., Vaughan, C.K., Meyer, P., Panaretou, B., Piper, P.W., Prodromou, C., Pearl, L.H., (2006) Nature, 440, pp. 1013-1017Stebbins, C.E., Russo, A.A., Schneider, C., Rosen, N., Hartl, F.U., Pavletich, N.P., (1997) Cell, 89, pp. 239-250Hohfeld, J., Cyr, D.M., Patterson, C., (2001) EMBO Rep., 2, pp. 885-890Pearl, L.H., Prodromou, C., (2002) Adv. Protein Chem., 59, pp. 157-186Bohen, S.P., (1998) Mol. Biol. Cell, 18, pp. 3330-3339Grad, I., McKee, T.A., Ludwig, S.M., Hoyle, G.W., Ruiz, P., Wurst, W., Floss, T., Picard, D., (2006) Mol. Cell. Biol., 26, pp. 8976-8983Toogun, O.A., Zeiger, W., Freeman, B.C., (2007) Proc. Natl. Acad. Sci. U.S.A., 104, pp. 5765-5770Woo, S.H., An, S., Lee, H.C., Jin, H.O., Seo, S.K., Yoo, D.H., Lee, K.H., Park, I.C., (2009) J. Biol. Chem., 284, pp. 30871-30880Tosoni, K., Costa, A., Sarno, S., (2011) Mol. Cell. Biochem., 356, pp. 245-254Poksay, K., Banwait, S., Crippen, D., Mao, X., Bredesen, D., Rao, R., (2011) J. Mol. Neurosci., pp. 1-12Patwardhan, C.A., Fauq, A., Peterson, L.B., Miller, C., Blagg, B.S.J., Chadli, A., (2013) J. Biol. Chem., 288, pp. 7313-7325Deleage, G., Combet, C., Blanchet, C., Geourjon, C., (2001) Comput. Biol. Med., 31, pp. 259-267Elble, R., (1992) Biotechniques, 13, pp. 18-20Oldenburg, K.R., Vo, K.T., Michaelis, S., Paddon, C., (1997) Nucleic Acids Res., 25, pp. 451-452Kroll, E.S., Hyland, K.M., Hieter, P., Li, J.J., (1996) Genetics, 143, pp. 95-102Kushnirov, V.V., (2000) Yeast, 16, pp. 857-860Corrêa, D.H.A., Ramos, C.H.I., (2009) Afr. J. Biochem. Res., 3, pp. 164-173Schuck, P., (2000) Biophys. J., 78, pp. 1606-1619Borges, J.C., Ramos, C.H.I., (2011) Curr. Med. Chem., 18, pp. 1276-1285Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J., Svergun, D.I., (2003) J. Appl. Crystallogr., 36, pp. 1277-1282Semenyuk, A.V., Svergun, D.I., (1991) J. Appl. Crystallogr., 24, pp. 537-540Svergun, D.I., (1992) J. Appl. Crystallogr., 25, pp. 495-503Blom, N., Gammeltoft, S., Brunak, S., (1999) J. Mol. Biol., 294, pp. 1351-1362Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., Mann, M., (2006) Cell, 127, pp. 635-648McLaughlin, S.H., Sobott, F., Yaol, Z.P., Zhang, W., Nielsen, P.R., Grossmann, J.G., Laue, E.D., Jackson, S.E., (2006) J. Mol. Biol., 356, pp. 746-758Karagoz, G.E., Duarte, A.M.S., Ippel, H., Uetrecht, C., Sinnige, T., Van Rosmalen, M., Hausmann, J., Rudiger, S.G.D., (2011) Proc. Natl. Acad. Sci. U.S.A., 108, pp. 580-585Trudeau, T., Nassar, R., Cumberworth, A., Wong, E.T., Woollard, G., Gsponer, J., (2013) Structure, 21, pp. 332-341Song, D., Li, L.S., Arsenault, P.R., Tan, Q., Bigham, A.W., Heaton-Johnson, K.J., Master, S.R., Lee, F.S., (2014) J. Biol. Chem.CHIR, JCB and LRSB are CNPq Research Fellows and thank FAPESP – Brazil (2011/23110-0; 2012/50161-8) and CNPq – Brazil for financial support. We thank the LNBio/CNPEM-ABTLuS (Campinas, Brazil) for making the AUC device available. We also thank the LNLS/CN

Conformational And Functional Studies Of A Cytosolic 90 kda Heat Shock Protein Hsp90 From Sugarcane.

Hsp90s are involved in several cellular processes, such as signaling, proteostasis, epigenetics, differentiation and stress defense. Although Hsp90s from different organisms are highly similar, they usually have small variations in conformation and function. Thus, the characterization of different Hsp90s is important to gain insight into the structure-function relationship that makes these chaperones key regulators in protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from sugarcane and its comparison with Hsp90s from other plants. Previous expressed sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the recombinant protein was purified and its conformation and function characterized. The structural conformation of Hsp90 was assessed by chemical cross-linking and hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays, which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong chaperone activity.6816-2

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved