Faculty & Research
Pathogenesis of African Trypanosomes/ Cellular-Signaling and Molecular Parasitology
African trypanosomiasis is caused by pathogenic protozoa that produce lethal infections in humans and livestock throughout sub-Saharan Africa. Over 60 million people in 36 countries are at risk of the disease with an estimated 300,000-500,000 deaths per year. In my lab, we study signal properties of Trypanosoma brucei. It is hypothesized that ablation of a signal or production of an inappropriate signal will be lethal to these organisms. While emphasis is placed upon the calcium regulatory network, our projects increasingly emphasize the signals and cytoskeletal elements responsible for cell division.
The research is greatly aided by the recently completed genome projects of the Tritryps (Trypanosoma brucei, Trypanosoma cruzi and Leishmania sp). Compared with the human host, the Trityps have notable deficiencies in their signal capabilities including the absence of trimeric G proteins, SH2 domains and serpentine receptors. In order to identify proteins that regulate cell division, inducible RNA interference and gene knockout are used. When the level of a putative regulatory protein is diminished, an evaluation of its role in cell cycle progression is made. Flow cytometry is used to analyze cell cycle progression. Yeast two-hybrid and endogenously expressed TAP-tags are used to identify binding partners within a pathway and laser scanning confocal microscopy is used to verify distribution of the candidate proteins during the cell cycle.
Recently we have used these methods to demonstrate that a RACK1 homologue (TRACK) from T. brucei is essential for completion of cytokinesis. In the absence of this homologue, trypanosomes initiate formation of a division furrow, but become stuck partway through the cleavage process. Rather than halt further cell division, the cells appear to lack appropriate checkpoints, and continue multiple rounds of partial cytokinesis. A movie of this novel phenotype is at: http://faculty.smu.edu/lruben/. We are testing two hypotheses in an effort to explain the ability of TRACK to regulate cytokinesis. The first hypothesis is that TRACK modulates signal events on polysomes and in this way affects translation efficiency of some cell cycle proteins. In support of this hypothesis, we have recently shown that TRACK is indeed a component of monosomes and polysomes. The second hypothesis is that TRACK brings signal proteins to the cleavage furrow. TRACK partners are being sought by yeast two-hybrid and TAP-tag methods.
Phenotype of trypanosomes after they are depleted of TRACK
Other projects examine a trypanosome Aurora kinase that we report is required for chromatin segregation, division of the nucleus, and coupling of kinetoplast replication to that of the nucleus. Kinase assays demonstrated an ability of trypanosome Aurora kinase-1 (TbAUK1) to phosphorylate recombinant histone H3 and H2B. We have used RNAi to knockdown Aurora kinase gene expression during the course of an infection in mice. Induction of RNAi with doxycycline caused the parasitemia to fall and allowed the mice to survive. These data provide proof of concept that the trypanosome Aurora kinase is a potential therapeutic target.
Because Aurora kinases play an important role in tuomorigenesis, the pharmaceutical industry has launched over 35 different programs aimed at developing anti-proliferative therapies against the Aurora kinases. The programs have advanced more than 18 different classes of small molecule Aurora inhibitors to pre-clinical, Phase I, and Phase II studies. With this large amount of chemical matter as a guide, my lab has begun a drug repurposing program aimed at identifying parent compounds and derivatives that can selectively target the trypanosome cell cycle. This latter project is in collaboration with medicinal chemists at Northeastern University. The trypanosome Aurora kinase-1 (TbAUK1) is different from the homologous human kinase at nine residues that are known to be responsible for drug binding. Analog series have been synthesized that explore the SAR for residues that interact with a hydrophobic pocket, hinge region or solvent space.
Ochiana, S.O., Pandarinath, V., Wang, Z., Kapoor, R., Ondrechen, M.J., Ruben, L., and Pollastri, M.P. (2012) The Human Aurora kinase inhibitor danusertib is a lead compound for anti-trypanosomal drug discovery via target repurposing. Eur. J. Med. Chem. [Epub ahead of print].
Jetton, N., Rothberg, K.G., Wise, J., Yan, L. Ball, H. and Ruben, L. (2009) The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms. Mol. Microbiol. 72, 442-458.
Rothberg, K. G., Burdette, Pfannstiel, J., D., Jetton, N., Singh, R. and Ruben, L. (2006) The RACK1 homologue from Trypanosoma brucei is required for onset and progression of cytokinesis. J. Biol. Chem. 281, 9781-9790.
Ruben, L., Kelly, J.M. and Chakrabarti, D. (2003) Cellular Signaling. In: Molecular and Medical Parasitology (Marr, J.J, Nilsen, T.W. and Komuniecki, R.,W. eds). pp. 241-276. Academic Press, UK
Ridgley, E; L. and Ruben, L. (2001) Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1 and sn-2 deacylation of phospholipids. Mol. Biochem. Parasitol. 114, 29-40.
Ridgley, E.L., Webster, P., Patton, C.L., and Ruben, L. (2000) Calmodulin-binding properties of the paraflagellar rod complex from Trypanosoma brucei Mol. Biochem. Parasitol. 109, 195-201.
Parsons, M. and Ruben, L. (2000) Pathways involved in environmental sensing in trypanosomatids. Parasitol. Today. 16, 58-64.
Ridgley, E.L., Xiong, Z.-H. and Ruben, L. (1999) Reactive oxygen species activate a calcium-dependent cell death pathway in Trypanosoma brucei. Biochem. J. 340, 33-40.
Eintracht, J., Maathai, R., Mellors, A. and Ruben, L. (1998) Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid. Biochem. J. 336, 659-666.
Xiong, Z.-H. and Ruben, L. (1998) Trypanosoma brucei: The dynamics of calcium movement between the cytosol, nucleus and mitochondrion of intact cells. Exp. Parasitol. 88, 231-239.
Xiong, Z.-H., Ridgley, E.L., Enis, D., Olness, F. and Ruben, L. (1997) Selective transfer of calcium from an acidic compartment to the mitochondrion of Trypanosoma brucei: Measurements with targeted aequorins. J. Biol. Chem. 272, 31022-31028.
Xiong, Z.-H. and Ruben, L. (1996) Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin. Mol. Biochem. Parasitol. 83, 57-67.
Ruben, L., Akins, C.D., Haghighat, N.G., and Xue, L. (1996) Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines. Mol. Biochem. Parasitol. 81, 191-200.Ridgley, E.L., Xiong, Z., Kaur, K.J. and Ruben, L. (1996) Genomic organization and expression of elongation factor-1 genes in Trypanosoma brucei. Mol. Biochem. Parasitol. 79, 119-123.
Wu, Y., DeFord, J., Benjamin, R., Lee, M.G.-S., and Ruben, L. (1994) The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei. Biochem. J. 304, 833-841.
Kaur, K. and Ruben, L. (1994) Protein translation elongation factor-1 from Trypanosoma brucei binds calmodulin. J. Biol. Chem. 269, 23045-23050.
Salmon, D., Geuskens, M., Hanocq, F., Hanocq-Quertier, J., Nolan, D., Ruben, L., and Pays, E. (1994) A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in Trypanosoma brucei. Cell 78, 75-86.
Wu, Y., Haghighat, N.G., and Ruben, L. (1992) The predominant calcimedins from Trypanosoma brucei comprise a family of flagellar EF-hand calcium-binding proteins. Biochem. J. 287, 187-193.
Haghighat, N.G. and Ruben, L. (1992) Purification of novel calcium-binding proteins from Trypanosoma brucei: properties of 22-, 24-, and 38-kilodalton proteins. Mol. Biochem. Parasitol. 51, 99-110.
Ruben, L. and Akins, C.D. (1992) Trypanosoma brucei: The tumor promoter thapsigargin stimulates calcium release from an intracellular compartment in slender bloodstream forms. Exp. Parasitol. 74, 332-339.
Ruben, L., Hutchinson, A., and Moehlman, J. (1991) Calcium homeostasis in Trypanosoma brucei: Identification of a pH sensitive non-mitochondrial calcium pool. J. Biol. Chem. 266, 24351-24358.
Ruben, L., Ridgley, E., Chan, E., and Haghighat, N.G. (1991) Variable surface glycoprotein from Trypanosoma brucei strain YTat1.1 contains a latent calmodulin-binding domain. Mol. Biochem. Parasitol. 46:123-136.Ruben, L., Haghighat, N., and Campbell, A. (1990) Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins. Exp. Parasitol. 70, 144-153.
OTHER LINKS TO TRYPANOSOMES
Food and Agriculture Organization of the UN
Awards and Recognition