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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:
In addition to being an important opportunistic pathogen, the protozoan parasite Toxoplasma gondii is an excellent model for the study of Apicomplexan parasites and pathogen-host interaction. The Sinai Laboratory has a long standing interest is understanding the mechanisms by which T. gondii manipulates host cell functions. These studies have led to several key findings regarding the parasite directed modulation of the host apoptotic pathway, cell cycle and more recently energy metabolism. Our work has further defined the molecular mechanisms employed by Toxoplasma to manipulate host NFkappaB activity as well as other signaling cascades. Most notably we have identified a parasite encoded I kappaB kinase activity (TgIKK) that localizes to the interface of the parasite and the host, the parasitophorous vacuole membrane (PVM). This work has been supported by an RO1 grant in its 11th year of funding that is currently in a no cost extension.
As the interface between the parasite and the host, the PVM represents a unique organelle that happens to be outside the body of the parent organism it shelters. We have recently completed a proteomic analysis of the PVM which has revealed number of new activities including a slew of hypothetical proteins several of which appear to the essential based on knock out studies. In addition we found proteins with homology to highly glycosylated proteophosphoglycans of Leishmania that we posit may be important in the development of the tissue cyst form of the parasite. Molecular studies of tissue cysts and parasite glycosylation pathways involved in their establishment are in their infancy. With this in mind we have initiated a detailed investigation into the glycosylation pathways of the parasite. This represents a new area of investigation in the laboratory. The project to establish the PVM proteome was supported by a completed R21 application. The characterization of several hypothetical ORF’s is supported by and active RO3 grant. There are currently 2 pending R21 applications to investigate the contribution of N-glycosylation in parasite biology and establish the contribution of the host glycosylation machinery to the modification of tissue cysts. An additional RO1 application to establish the contribution of several hypothetical ORF’s to the formation and stability of the tissue cyst is in early stages of preparation.
In the course of studies to identify the gene for TgIKK, we stumbled upon a homolog of the yeast autophagy regulator ATG1. This has prompted the investigation of autophagic pathways in the parasite. Our data indicate that autophagy represents a cell death pathway in Toxoplasma and may be important in understanding the cytocidal effects of drugs. In addition we find that both TgATG1 and TgATG8 have functions outside the autophagic pathway which we are now in the process of dissecting. A comprehensive RO1 application will be submitted soon once the initial papers are in press. This proposal will be aimed at dissecting autophagy in Toxoplasma and the role of TgATG1 in parasite biology.
In addition to these research foci, we are actively involved in several collaborative projects, Most notable among these is a collaboration with Dr. Beth Garvy (Dept. of MIMG, UK) to establish purification methods for Pneumocystis and develop platforms for proteomic analysis. The proteomic work is done in collaboration with Dr. Bert Lynn (Dept. of Chemistry, UK), with whom we are developing methodological approaches for sample preparation of “difficult” low abundance material. We have also established collaborations with Dr. Paul Roepe (Georgetown University) to examine autophagic mechanism in malaria and with Dr. Jim Ajioka (Cambridge University) to investigate parasite induced changes in host energy metabolism and glucose flux. Finally we are assisting Dr. Srinath Kamineni (Dept of Surgery, UK) with the development of proteomic approaches to assess collagen isoform diversity in traumatized elbow capsules. A grant in support of this work is currently pending.
Grants:
Active Grants
12-14 NIH/NIAID R21AI098371-01A1 Contribution of N-glycosylation to the Toxoplasma glycoproteome
Role: PI: 10% effort ($275,000 direct cost).
10-12 NIH/NIAID, RO3AI092170-02, Novel Activities and the T. gondii vacuolar membrane.
Role: PI: 3% effort, ($100,000- direct cost).
Pending Grants
12-14 NIH/NIAID R21AI099509-01 Host glycosyltransferases in the glycosylation of Toxoplasma proteins.
Role: PI: Sinai ($275,000 direct cost).
12-13 KSEF-2624-RDE-015 (Kentucky Science and Engineering Foundation). Autophagy in the protozoan parasite Toxoplasma gondii.
Role: PI: ($50,000 direct cost).
12-17 NIH/NIAID-RO1 AI045957-12 The physiology of drug resistant malaria
Role: co-Investigator (10% effort) PI: Paul Roepe, Georgetown University.
Selected Recent Publications:
Ghosh D, J.L. Walton, P.D. Roepe and AP Sinai (2012) Establishment of autophagy as a cell death mechanism in Toxoplasma gondii. Cell Microbiol 14(4): 589-607 PDF
Sinai AP, ES Kaneshiro, H. Ward, LM Weiss and MT Cushion (2012) The state of research for AIDS-associated opportunistic infections: a call the research communities and funding agencies. Euk. Cell 11(2): 90-27 PDF
Carmen, JC. and AP Sinai (2011) The differential effect of Toxoplasma gondii infection on the stability of BCL2-family members involves multiple activities.
Front. Microbio. doi: 10.3389/fmicb.2011.00001 PDF
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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