Molecular Biology Faculty
Ileana M. Cristea
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 (609) 258-9417 |
 Lewis Thomas Lab, 210 |
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Lab (609) 258-9425 |
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Faculty Assistant
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Ellen Brindle-Clark
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(609) 258-541 |
Cristea Research LabResearch Focus
Proteomic-Genomic approaches to chromatin and its modulation by viruses
The research in my laboratory is aimed at understanding mechanisms that control the fate of cells under invasion by pathogens, with particular emphasis on the viral modulation of chromatin remodeling machineries. The wide range of diseases caused by viruses, from the common cold to arthritis to AIDS, is a reflection of the diverse ways in which viruses interact with host cells and manipulate their basic functions. How do viruses accomplish these tasks, and how do cells respond and adapt to infection? In approaching this question, we use the full power of differential genomics and proteomics to study the modulation of chromatin remodeling complexes in both normal and virally infected cells. The state-of-the-art mass spectrometry in my laboratory focuses on the use of novel instrument configurations, which are ideally suited for the fast and sensitive characterization of macromolecular complexes.
Exploring the temporal and spatial dynamics of protein interactions
Protein interactions, of a stable or transient nature, underlie the majority of cellular processes. Knowledge of the localization, composition, and organization of protein complexes at a particular point in space and time provides key insights into their functions. We have developed a strategy that uses a single fluorescent tag to both visualize proteins in live cells and capture their interacting macromolecular partners via high-affinity immunopurifications. My previous work showed that rapid isolations are essential to preserving protein complexes close to their original state in the cell. Accordingly, we use cryogenic cell lysis in combination with 5-60 minutes immunoisolations on magnetic beads coated with in-house developed antibodies. We found that this methodology opens new doors for almost every protein complex that we have approached, leading to both insights into cellular processes and new questions. However, gaining access to low-stoichiometry and short-lived interactions is still challenging, and further methodological improvements are a necessity. The work in my laboratory will seek to push the boundaries of current proteomic techniques, and continue to devise new tools for exploring protein interactions. Effort will be placed into further decreasing the time necessary to go from a live cell to an isolated protein complex.
Walking in the proteomic footprints of viral infection
By making use of finely tuned protein interactions, viruses succeed in sabotaging intricate host processes and turning cellular systems to their own use. Although virus-host interactions have been extensively studied, our knowledge of the detailed interactions between viral and host proteins during the course of infection remains quite limited. I was interested in approaching the dynamic viral infection by tracking the localization and interactions of viral proteins in host systems as a function of infection time. We have successfully implemented our new methodologies to uncover the viral-host protein interactomes of the mosquito-borne Alphavirus Sindbis, HIV-1, and, most recently, the human cytomegalovirus. While our findings led to new hypotheses as to how viruses manipulate host cellular processes, the richness of the novel identified interactions reminded us that we are still in virtually uncharted territory in understanding the complexity of the viral-host protein relationship.
Understanding the role of chromatin remodeling complexes during viral infection
In all eukaryotes, gene expression is regulated by chromatin remodeling complexes, which use an intricate system of specialized enzymes to coordinate a series of posttranslational modifications on the N-terminal tails of histones. Our preliminary studies indicated that viruses may modulate gene expression through interactions with chromatin remodeling complexes. Indeed, histone deacetylases are targeted during infection by numerous viruses, and are reported to mediate the latency stages of cells infected with HSV-1 and HIV. Our approach provides a close look at this relationship, showing that HIV, HCMV, and Sindbis viral proteins recruit members of the chromatin remodeling complexes, including histone deacetylases, acetyltransferases, demethylases, and methyltransferases, as well as histone chaperones. While such interactions are in part virus-specific, some may reveal common pathways utilized by viruses to determine the outcome of the infection or by the cell in response to injury.
Characterizing chromatin remodeling complexes in normal and virally infected cells
Now that we have robust and efficient methodologies for the study of macromolecular interactions, we have initiated a comprehensive mapping of the chromatin remodeling interactome in normal cells. The novel interacting partners of HDAC1 so far uncovered are indicative of the multi-faceted roles of HDAC complexes in the precise orchestration of histone modifications, and we are currently exploring the roles of these specific interactions. Additionally, these analyses of unperturbed cells provide a baseline for further studies of the way cells respond to changing conditions. In particular, we will examine the effects of viral infection on chromatin remodeling complexes using approaches such as 1) tracking the localization of members of the chromatin remodeling complexes, 2) revealing the proteins that assemble these complexes, 3) identifying changes in protein-DNA interactions, 4) mapping posttranslational modifications on chromatin remodeling complexes and their targets, 5) determining protein turnover using mass spectrometry, and 6) elucidating the structures of chromatin remodeling assemblies using proteomics-based methodologies.
The proteomic approaches employed in our laboratory add a powerful new dimension to the already considerable reach of molecular biology in querying nature. Employing new ways to look at long-standing questions is one of the strongest leitmotifs in science, giving us new opportunities to appreciate, as Darwin saw it, the "grandeur in this view of life."
Selected Publications
Kramer T, Greco TM, Taylor MP, Ambrosini AE, Cristea IM, Enquist LW. (2012) Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons. Cell Host Microbe.12: 806-14. Pubmed
Miteva YV, Budayeva HG, Cristea IM. (2012) Proteomics-based methods for discovery, quantification, and validation of protein-protein interactions. Anal Chem. 85: 749-768. Pubmed
Greco TM, Miteva Y, Conlon FL, Cristea IM. (2012) Complementary proteomic analysis of protein complexes. Methods Mol Biol. 917: 391-407. Pubmed
Guise AJ, Greco TM, Zhang IY, Yu F, Cristea IM. (2012) Aurora B-dependent regulation of class IIa histone deacetylases by mitotic nuclear localization signal phosphorylation. Mol Cell Proteomics. 11: 1220-9. Pubmed
Gudleski-O'Regan N, Greco TM, Cristea IM, Shenk T. (2012) Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity. Cell Host Microbe. 12: 86-96. Pubmed
Li T, Diner BA, Chen J, Cristea IM. (2012) Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc Natl Acad Sci. 109: 10558-10563. PubMed
Tsai YC, Greco TM, Boonmee A, Miteva Y, Cristea IM (2012) Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Mol Cell Proteomics. 11: 60-76. PubMed
Sabouri N, McDonald KR, Webb CJ, Cristea IM, Zakian VA. (2012) DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase. Genes Dev. 26: 581-593. PubMed
Goodarzi H, Najafabadi HS, Oikonomou P, Greco TM, Fish L, Salavati R, Cristea IM, Tavazoie S (2012) Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Nature. 485: 264-268. PubMed
Cowles CE, Li Y, Semmelhack MF, Cristea IM, Silhavy TJ. (2011) The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli. Mol Microbiol. 79: 1168-1181. PubMed
Kramer T, Greco TM, Enquist LW, Cristea IM. J Virol. (2011) Proteomic characterization of Pseudorabies virus extracellular virions. J Virol. 85: 6427-6441. PubMed
Blanco MA, Aleckovic M, Hua Y, Li T, Wei Y, Xu Z, Cristea IM, Kang Y. (2011) Identification of Staphylococcal nuclease domain containing 1 (SND1) as a Metadherin-interacting protein with metastasis-promoting functions. J Biol Chem. 286: 19982-19992. PubMed
Cowles CE, Li Y, Semmelhack MF, Cristea IM, Silhavy TJ. (2011) The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli. Mol Microbiol. 79: 1168-1181. PubMed
Greco TM, Yu F, Guise AJ, Cristea IM. (2010) Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation. Mol Cell Proteomics. 10: M110.004317. PubMed
Moorman NJ, Sharon-Friling R, Shenk T, Cristea IM. (2010) A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation. Mol Cell Proteomics. 9: 851-860. PubMed
Terhune SS, Moorman NJ, Cristea IM, Savaryn JP, Cuevas-Bennett C, Rout MP, Chait BT, Shenk T. (2010) Human cytomegalovirus UL29/28 protein interacts with components of the NuRD complex which promote accumulation of immediate-early RNA. PLoS Pathog. 6: e1000965. PubMed
Cristea IM, Moorman NJ, Terhune SS, Cuevas CD, O'Keefe ES, Rout MP, Chait BT, Shenk T. (2010) Human cytomegalovirus pUL83 stimulates activity of the viral immediate-early promoter through its interaction with the cellular IFI16 protein. J Virol. 84: 7803-7814. PubMed
Carabetta VJ, Silhavy TJ, Cristea IM. (2010) The response regulator SprE (RssB) is required for maintaining PAP I-degradosome association during stationary phase. J Bacteriol. 192: 3713-3721. PubMedCristea IM, Rozjabek H, Molloy KR, Karki S, White LL, Rice CM, Rout MP, Chait BT, Macdonald MR. (2010) Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: a role for G3BP1 and G3BP2 in virus replication. J Virol. 84: 6720-6732. PubMed
Carabetta VJ, Li T, Shakya A, Greco TM, Cristea IM. (2010) Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions. J Am Soc Mass Spectrom. 21: 1050-1060. PubMedGoldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Dewell S, Law M, Guo X, Li X, Wen D, Chapgier A, DeKelver RC, Miller JC, Lee YL, Boydston EA, Holmes MC, Gregory PD, Greally JM, Rafii S, Yang C, Scambler PJ, Garrick D, Gibbons RJ, Higgs DR, Cristea IM, Urnov FD, Zheng D, Allis CD. (2010) Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140: 678-691. PubMedMoorman NJ, Sharon-Friling R, Shenk T, Cristea IM. (2009) A targeted spatial-temporal proteomic approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation. Mol Cell Proteomics. 9: 851-860. PubMed
Luo Y, Li T, Yu F, Kramer T, Cristea IM. (2009) Resolving the composition of protein complexes using a MALDI LTQ orbitrap. J Am Soc Mass Spectrom. 21: 34-46. PubMed
Selimi F, Cristea IM, Heller E, Chait BT, Heintz N. (2009) Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse. PLoS Biol 7: 948-957. PubMed
Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T. (2008) Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Cell Host Microbe 3: 253-262. PubMed
Glavy JS, Krutchinsky A, Cristea IM, Berke IC, Boehmer T, Blobel G, Chait BT (2007). Cell-cCycle dependent phosphorylation of the nuclear pore Nup107-160 subcomplex. Proc Natl Acad Sci USA.104: 3811-3816. PubMed
Warwood S, Mohammed S, Cristea IM, Evans C, Whetton AD, Gaskell SJ (2006). Guanidination chemistry for qualitative and quantitative proteomics. Rapid Commun Mass Spectrom 20: 3245-3256. PubMed
Cristea IM, Carroll JW, Rout MP, Rice CM, Chait BT, MacDonald MR (2006) Tracking and elucidating alphavirus-host protein interactions. J Biol Chem 281: 30269-30278. PubMed
Wang QJ, Ding Y, Kohtz DS, Mizushima N, Cristea IM, Rout MP, Chait BT, Zhong Y, Heintz N, Yue Z (2006). Induction of autophagy in axonal dystrophy and degeneration. J Neurosci 26: 8057-8068. PubMed
Cristea IM, Williams R, Chait BT, Rout MP (2005). Fluorescent proteins as proteomic probes. Mol Cell Proteomics 4: 1933-1941. PubMed
Cristea IM, Degli Esposti M (2004) Membrane lipids and cell death: an overview. Chem Phys Lipids 129: 133-160. PubMed
Sorice M, Circella A, Cristea IM, Garofalo T, Di Renzo L, Alessandri C, Valesini G, Degli Esposti M (2004). Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. Cell Death Differ 11: 1133-1145. PubMed
Meneses-lorente G, Guest P, Lawrence J, Muniappa N, Knowles M, Skynner H, Salim K, Cristea I, Mortishire-Smith R, Gaskell SJ, Watt A (2004). A proteomic investigation of drug induced steatosis in rat liver. Chem Res Toxicol 17: 205-212.
Cristea IM, Gaskell SJ, Whetton DW (2004). Proteomics techniques and their application to hematology. Blood 103: 3624-3634. PubMed
Degli Esposti M, Cristea IM, Gaskell SJ, Nakao Y, Dive C (2003) Pro-apoptotic Bid binds to monolysocardiolipin, a new molecular connection between mitochondrial membranes and cell death. Cell Death Differ 10: 1300-1309. PubMed
Fairlamb IJS, Dickinson JM, Cristea IM (2001) Influence of palladium(II) complexes on the cycloaddition of α-bromoalkyl ketenes to cyclopentadiene. Tetrahedron 57: 2237.
Fairlamb IJS, Dickinson JM, Cristea IM (2000) Palladium-catalysed [π2a + π2s] of α-bromoalkyl ketenes to cyclopentadiene. Tetrahedron Letters 41: 3739.
