As the site of secretory and membrane protein biogenesis, the endoplasmic reticulum (ER) performs numerous protein biosynthetic functions essential for eukaryotic life. As the site for secretory and membrane protein biogenesis, the ER a represents the entry point to the secretory pathway. Coupled to this is a key function in protein folding and assembly, where nascent proteins interact with ER resident molecular chaperones to achieve their native structure and activity. These two aspects of ER function are integrated in the unfolded protein response pathway (UPR). The UPR provides a mechanism for cells to respond to the potentially pathological problem of unfolded protein accumulation, via regulation of the transcriptional and translational programs of the cell. It is now known that the UPR serves critical roles in the response to cell stress, and in normal cell growth and differentiation.
   My laboratory has focused its research efforts on two topics: understanding how eukaryotic cells compartmentalize and regulate the protein synthesis activities of the cytosol and ER, and identifying the cellular, tissue, organ and organismal functions of the ER chaperone GRP94, the ER paralog of Hsp90, whose expression is essential for, and restricted to, multicellular eukaryotes. Regarding the former, we have discovered that ER-bound serve a key role in cellular protein synthesis and may represent the primary protein synthesis compartment in the cell, for secretory, integral and cytosolic proteins alike. This hypothesis extends from the findings that mRNAs encoding cytosolic and scretory/membrane proteins can be localized to the ER, for translation on membrane-bound ribosomes, and that steady state protein synthesis on the ER is substantially higher than protein synthesis in the cytosol. This latter phenomenon appears to reflect a compartmentalization of many of the reactions of protein synthesis . From these initial findings, we are working to understand the rules that govern ribosome and mRNA partitioning between the cytosol and ER of higher eukaryotic cells. These studies identify a new paradigm for defining the signals that govern the localization and trafficking dynamics of ribosomes and mRNA molecules in the cell.
   As a component of our research on protein translocation/protein synthesis, we have initiated studies on the regulation of ribosome and mRNA trafficking on the ER membrane during cell stress and recovery. Cells subjected to stress conditions such as oxygen or nutrient deprivation undergo a stress response in which global protein synthesis is attenuated and resident ER chaperone synthesis activated. What cellular mechanisms are responsible for maintaining ER chaperone synthesis and translocation when global protein synthesis is inhibited? The overall aim of these studies is to develop an integrated physiological perspective on the regulation of ribosome and mRNA trafficking during cell stress and recovery.
   Molecular chaperones are a family of (poly)peptide binding proteins that assist in protein folding and assembly. The ER harbors an array of chaperones including GRP170, GRP94, the ER Hsp90 chaperone, GRP78(BiP), an ER Hsp70 chaperone, protein disulfide isomerase, calreticulin and others. In a fascinating and unanticipated development, GRP94 (gp96, ERp99, endoplasmin), has been also demonstrated to function as tumor rejection antigen. Thus, when isolated from tumor tissue, GRP94, can function as an immunotherapeutic vaccine against its tumor tissue of origin. What is the mechanism by which GRP94 can elicit cellular immune responses? To address this primary question, we are utilizing biochemical, cell biological and immunological methods to define the regulation of GRP94 (poly)peptide binding activity, the recognition and processing of GRP94 by immune effector cells and the molecular mechanism of GRP94-dependent tumor rejection.
   Though almost all chaperones have orthologs throughout the Eubacteria and the Eukarya, GRP94 has a very limited distribution, and is found only in animals (the metazoans) and higher (multicellular) plants. These findings suggest that the cohort of proteins that require GRP94 for functional expression play critical and in many cases essential roles in the cell-cell communication/cell-cell interaction processes that allow cells to function as tissues. If this hypothesis is correct, then the GRP94 "proteome" contains many of the secrets surrounding the cellular origin of multicellular life. To test this hypothesis, we are using Drosophila as a model system to identify the GRP94 proteome and to determine how this proteome functions to enable multicellularity and control tissue morphogenesis. In this regard, it is intriguing to note that mammalian tissue cultures do not need GRP94 for viability, yet the GRP94 knockout in mouse is embryonic-lethal.

E-mail
c.nicchitta@cellbio.duke.edu

436A Nanaline Duke Bldg., Box 3709
Duke University Medical Center
Durham, NC 27710

Telephone
919-684-8948 (office)
919-684-8980 (lab)
Fax 919-684-5481

New Postdoc Position Available

Loss of Gp93 function in the eye imaginal disc via EGUF/hid or RNAi results in disruptions in eye development. Gp93r was expressed in the eye imaginal disc via the EGUF/hid method. Scanning electron micrographs of eyes are depicted, with panels A and B representing the parent FRT82B line and panels E and F, the Gp93r line. The bar in panels A and E = 200 mm; in panels B and F = 100 micron. Panels C,D,G,H depict scanning electron micrographs of UAS-Gp93 dsRNA (panels C, D) and eyeless GAL4/UAS-Gp93-dsRNA (panels G, H). The bar in panels C and G = 200 micron; in panels D and H = 100 micron.

Selected Publications
Pyhtila B, Zheng T, Lager PJ, Keene JD, Reedy MC, Nicchitta CV. (2008) Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum. RNA. 14(3):445-53. -PDF-

Stephens SB, Nicchitta CV. (2008) Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells. Mol Biol Cell. 19(2):623-32. -PDF-

Yewdell JW, Nicchitta CV. (2006) The DRiP hypothesis decennial: support, controversy, refinement and extension. Trends Immunol. 2006 27(8):368-73. -PDF-

Chu F, Maynard JC, Chiosis G, Nicchitta CV, Burlingame AL. (2006) Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94. Protein Sci. 15(6):1260-9.

Lerner RS, Nicchitta CV. (2006) mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation. RNA. 12(5):775-789. -PDF-

Stephens, S.B., Dodd, R.D., Brewer, J.W., Lager, P.J., Keene, J.D., and Nicchitta, CV. (2005) Stable Ribosome Binding to the Endoplasmic Reticulum Enables Compartment-Specific Regulation of mRNA Translation. Mol. Biol. Cell 16(12):5819-31. -PDF-

Nicchitta, CV., Lerner, R.S., Stephens, S.B., Dodd, R.D., and Pyhtila, B. (2005) Pathways for compartmentalizing protein synthesis in eukaryotic cells: The Template Partitioning Model. Mol. Cell. Biochem. 83(6):687-695. -PDF-

Baker-LePain JC, Sarzotti M, Nicchitta CV. (2004) Glucose-regulated protein 94/glycoprotein 96 elicits bystander activation of CD4(+) T cell Th1 cytokine production in vivo. J Immunol. 2004;172(7):4195-203.

Rosser MF, Trotta BM, Marshall MR, Berwin B, Nicchitta CV. (2004) Adenosine nucleotides and the regulation of GRP94-client protein interactions. Biochemistry. 43(27):8835-45.

Berwin B, Hart JP, Rice S, Gass C, Pizzo SV, Post SR, Nicchitta CV. (2003) Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells. EMBO J. 2003 22(22):6127-36. -PDF-

Soldano KL, Jivan A, Nicchitta CV, Gewirth DT. (2003) Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation. J Biol Chem. 278(48):48330-8.

Lerner, R.S., Seiser, R.M., Zheng, T., Lager, P.J., Reedy, M.C., Keene, J.D., Nicchitta, C.V. (2003) Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes. RNA. 9(9):1123-37.

Current Projects
Protein synthesis regulation:
  What role does protein synthesis play in the regulation of ribosome binding and release on the ER?
  How is the protein synthesis activity of ER-bound ribosomes regulated during normal and stress conditions?
  What is the mechanism of mRNA targeting to, and release from, the ER membrane?
  Do (subclasses of) mRNAs contain ER-directed localization information?

Chaperone Function:
  How are GRP94 interactions with polypeptide substrates regulated?
  What is the identity of the GRP94 "proteome"? (those proteins whose functional expression requires GRP94).
  How is GRP94 recognized by the cells of the immune system?