Different strategies for proteomic identification of SUMOylated proteins in Trypanosoma brucei

PAULA A. IRIBARREN; MARÍA A. BERAZATEGUI; JUAN J. CAZZULO; JULIO C. BAYONA; VANINA E. ALVAREZ

Buenos Aires

Congreso; Ubiquitin and UBLs. At the crossroads from chromatin to protein; 2014

EMBO-CONICET

Different strategies for proteomic identification of SUMOylated proteins in Trypanosoma brucei Paula A. Iribarren, María A. Berazategui, Juan J. Cazzulo, Julio C. Bayona and Vanina E. Alvarez Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús ?Dr. Rodolfo A. Ugalde? IIB-INTECH / UNSAM-CONICET Argentina Trypanosomatids are protozoa responsible for human diseases affecting a high number of people. In Trypanosoma brucei, the etiological agent of african trypanosomiasis, SUMO resulted essential for procyclic (PC)1 and bloodstream (BS)2 forms. There is also strong evidence that supports the association of SUMO with the expression of the variable surface glycoprotein, responsible for the evasion of the immune response (López Farfán D et al., manuscript under revision) The aim of this work is to compare different strategies for proteomic identification of SUMO conjugates in T. brucei. With this purpose we have generated different cell lines with SUMO chromosomal tagging that enable SUMO expression at physiological levels and provide at the same time tags (His and HA tags) for tandem affinity purification of the conjugates. We have obtained parasites with a single and a double replacement of the SUMO alleles, achieving a significant improvement in the yield of the purification of conjugates when comparing it to the one obtained with SUMO overexpression cell lines3. Proteomic analysis of these samples, obtained either by 1D LC MS/MS or 2D LC MS/MS, revealed that contaminants still represented a major issue as the proteins present in the samples did not differed significantly from the proteins present in a mock purification from a wild-type cell line performed as control. We therefore thought to apply a complementary approach to reduce unspecific purification of contaminant proteins by a LysC-based strategy as described by Matic et al.4. Briefly, using a Lysine-deficient His-HA SUMO cell line and digesting the samples with Lys-C it is possible to degrade all the proteins present in the sample with the exception of SUMO, that remains covalently attached to the LysC-digested target. At the same time, the introduction of an arginine residue before the GG motif is supposed to map the acceptor lysines for SUMO in the endogenous substrate. Considering the potential overlap between ubiquitin and SUMO targets, we are currently working on a new scheme, developed by Ronald Hay´s group (personal communication), based on a first Ni-NTA affinity chromatography followed by a second purification step based on the presence of the GG motif in the lysine of the protein that is being modified. This approach would allow the identification of SUMOylated proteins and to unambiguously identify the acceptor Lysine. 1. Liao S., Wang T., Fan K. and TU X. (2010) The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei. Nature. 414:759-763. 2. Obado S. O., Bot C., Echeverry M. C., Bayona J. C., Alvarez V. E., Taylor M. C. and Kelly J. M. (2011) Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei. Nucleic Acids Res. Feb; 39 (3): 1023-1033 3. Bayona, J. C., Nakayasu, E. S., Laverriere, M., Aguilar, C., Sobreira, T. J., Choi, H., Nesvizhskii, A. I., Almeida, I. C., Cazzulo, J. J. and Alvarez, V. E. (2011) SUMOylation pathway in Trypanosoma cruzi: functional characterization and proteomic analysis of target proteins. Mol Cell Proteomics 2011 Dec;10(12):M110.007369. 4. Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, Gnad F, Mann M, Vertegaal AC. (2010). Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell 39, 641-52.