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Office: (859) 323-6680
Fax: (859) 257-8994
Lab: (859) 323-1341
Email: sinai@uky.edu
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Anthony Sinai, Ph.D.
Associate Professor
Doctoral Studies: University of Rochester.
Postdoctoral: Yale University.
Research Statement:
Intracellular pathogens like the protozoan parasite Toxoplasma gondii subvert host cell functions to establish a successful infection. We are particularly interested in understanding how parasites manipulate the NFkappaB pathway as we have shown that activation of this cascade is required for the parasite induced blockade of host cell apoptosis. A central player in the activation of NFkappaB by the parasite is a parasite derived kinase activity termed TgIKK that is localized to the vacuolar membrane surrounding the intracellular parasite. TgIKK activity appears directed at the host inhibitor of NFkappaB protein (IkappaB) targeting residues critical for NFkappaB activation. The activation of NFkappaB by the parasite is multifactorial as mutants with diverse lesions (in both known and hypothetical loci) have been isolated using a flow cytometry based genetic screen. Investigations into the blockade of apoptosis and NFkappaB activation have lead to investigations in other cellular functions including the impact of infection on the host cell cycle and energetics. We are beginning to investigate whether these changes impact parasite physiology and may serve as critical cues in differentiation into the relatively dormant cyst forms.
As the interface between the parasite and the host cell, the parasitophorous vacuole membrane (PVM) serves as a platform for diverse activities in the pathogen-host cell interaction. Investigation into PVM biology has been limited due to its being present only in infected cells. In addition the intimate association of the PVM with host organelles that precludes its purification for biochemical analysis. We developed a pair of novel antibody reagents against the PVM-fraction and used them to identify novel PVM-localized activities. This study relied on the development of sample preparation techniques and state of the art proteomics (in collaboration with Dr. Bert Lynn, Department of Chemistry) and has resulted in the provisional identification of 70 novel proteins. Several of these targets have been confirmed and include a PVM –localized E2- Ubiquitin ligase (potentially affecting NFkappaB signaling) and a family of predicted proteophosphoglycans (PPG’s) that one may expect in a Toxoplasma cyst wall. The detection of several PPG’s at the PVM of RH strain parasites that typically do not form cysts was truly surprising. The PVM proteome presents an excellent opportunity to analyze diverse and interesting parasite proteins and reveal the complexity of the Toxoplasma-host cell interaction.
By combining state of the art cell biological, biochemical and increasingly molecular biological approaches to dissect the mechanisms underlying the disruption of cellular function by Toxoplasma we hope to gain new insights into parasite biology as well as broader questions on the microbial pathogenesis.
Selected Recent Publications:
Carmen, JC, RC Southard and AP Sinai (2008) The complexity of signaling in host-pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation. Exp. Cell Res.314: 3724-36 PDF
Liu T., AM Martin, AP Sinai and BC Lynn (2008) Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis. J. Proteome Res. 7(10): 4256-65 PDF
Sinai AP (2008) Biogenesis of and activities at the Toxoplasma gondii parasitophorous vacuole membrane. Subcell Biochem. 47: 155-64 (invitedreview). , PDF
Molestina RE, N El-Guendy and AP Sinai (2008) Infection with Toxoplasma gondii results in dysregulation of the host cell cycle. Cell Microbiol. 10(5): 1153-65 PDF
Martin, AM, T Liu, BC Lynn and AP Sinai (2007) Elimination of affinity reagent interference for the mass spectrometric detection of low abundance proteins following immunoprecipitation. J. Proteome Res. 6(12): 4758-62 PDF
Carmen JC and AP Sinai (2007) Suicide Prevention: Disruption of apoptotic pathways by protozoan parasites. Molec. Microbiol. 64(4): 904-16 (invited review). , PDF
Martin A.M., T. Liu, B.C. Lynn and AP Sinai (2007). The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border. J. Euk. Microbiol. 54(1): 66-72 PDF
Carmen JC, L Hardi and AP Sinai (2006) Toxoplasma gondii inhibits UV-induced apoptosis at multiple points along the mitochondrion-dependent pathway. Cell. Microbiol. 8(2): 301-15 PDF
Molestina RE and AP Sinai (2005) Requirement for the integrity of the host IKK signalosome and a Toxoplasma gondii–derived IKK activity in the parasite-directed modulation of NFkB. J. Cell Sci. 118(24): 5785-96. PDF
Molestina R.E and AP Sinai (2005) A novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkBa. Cell. Microbiol. 7(3): 351-62 (cover of journal) PDF
Molestina R.E., T.M. Payne, Isabelle Coppens and A.P. Sinai (2003). Activation of NF-kB by Toxoplasma gondii correlates with increased expression of anti-apoptotic genes and localization of phosphorylated IkB to the parasitophorous vacuole membrane. J. Cell Sci. 116(21): 4359-71 (highlighted in journal) PDF
Payne, T.M., R.E. Molestina and A.P. Sinai (2003) Inhibition of caspase activation and a requirement for NF-kB function in the Toxoplasma gondii mediated blockade of host apoptosis.J. Cell Sci.116(21): 4345-58. (highlighted in journal) PDF
Sinai A. P. and K.A. Joiner (2001), The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membraneJ. Cell Biol. 154: 95-108 ( highlighted in journal)
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Sinai A.P., P. Webster and K.A. Joiner (1997), Association of host endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J. Cell Sci. 110: 2117-28. PDF
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