Visualization of Protein Ubiquitination During T Cell Development In Vivo

Deyu Fang, Ph.D.

University of Missouri-Columbia

Funded in June, 2006: $250000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Visualizing Metabolic Processes Essential to Effective Immune T Cell Development

Researchers will use cellular imaging in mice to see how two proteins, which are essential for the early stages of immune T cell development, then degrade before they inhibit the T cells’ further growth and maturation.  Understanding this normal degradation process may provide important insight into how failure of these proteins to degrade at the proper time can lead to malfunctioning T cells that attack the body’s own tissues.

Immune T cells learn to recognize and attack specific invaders.  Occasionally, during T cell development, they learn incorrectly.  They mistake the body’s own tissues as foreign and attack them, producing autoimmune diseases. In the early stages of T cell development, two proteins—PU.1 and E2A—are essential.  These two proteins, whose actions are tightly controlled, must then completely disappear at later stages of T cell development.  Otherwise, for instance, PU.1 will block T-cell maturation, which will result in immune deficiency.  The researchers hypothesize that the two proteins clear out through a process of metabolic degradation called “ubiquitination.” Improper regulation of the degradation of either protein, according to the researchers’ hypothesis, alters T cell development and causes autoimmune diseases such as arthritis and juvenile diabetes.

The researchers aim to determine the molecular process of PU.1 and E2A degradation during T cell development by using cellular imaging to visualize the process in mice.  They will image the two proteins at different stages of T cell development, using “ubiquitin-mediated fluorescence complementation” in single living cells.  By revealing the PU.1 and E2A degradation process that promotes late stage T cell development, the investigators can identify molecular targets for drugs that might prevent malfunctions in this process that produce autoimmune diseases.     

Significance:  This study of metabolic processes that are critical for normal immune T cell development may lead to development of new types of therapies to treat T cell malfunctions that produce autoimmune diseases.

ABSTRACT

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Visualization of Protein Ubiquitination During T Cell Development In Vivo

The transcription factors PU.1 and E2A play important roles in T-cell development. Their expression levels are tightly controlled during the development of immune cells. Dysregulated transcription activation of PU.1 and E2A alters T-cell development and causes immune diseases, such as diabetes and arthritis. The up-regulation of these proteins is controlled at the transcriptional level. The mechanisms of how these proteins are down-regulated at certain stages of T-cell development, however, remain unknown. We hypothesize that ubiquitination eliminates transcription factors PU.1 and E2A to promote T-cell development. We propose to visualize PU.1 and E2A ubiquitination in the primary T cells at different developmental stages in mice. The development of ubiquitin-mediated fluorescence complementation (UbFC) assay allows imaging proteins modified by ubiquitin-family peptides in single living cell, thus overcomes the difficulties for ubiquitination analysis due to the limited cell numbers of immature T cells at certain developmental stages.

First we will visualize PU.1 and E2A ubiquitination in vitro to elucidate how ubiquitination regulates 1) subcellular localization, 2) protein stability, 3) the nuclear-cytoplasm shuttling and 4) transcription activation of PU.1 and E2A in living cells. Next, we will visualize PU.1 and E2A ubiquitination in mice. To this aim, we will infect mice bone marrow cells (immune stem cell) with lentvirus that carry a fusion protein of the N-terminus of YFP with ubiquitin (YN-Ub) and PU.1/E2A-YC (the C-terminus of YFP) expression genes and adoptively transfer back to mice, and then visualize PU.1/E2A ubiquitination during T cells development in vivo. These experiments will illuminate at which developmental stage PU.1 and E2A is ubiquitinated and how their ubiquitination is kinetically regulated during T cell development. These findings will provide a directly evidence for ubiquitination in regulating T-cell development in vivo. Finally, we will use YN-Ub and PU.1-YC or E2A-YC transgenic mice to investigate the involvement of ubiquitination of PU.1 and E2A in the development of collagen-induced arthritis in mice.

The results from our proposed studies will illuminate how protein ubiquitination controls the protein levels of PU.1 and E2A to regulate T-cell development. These findings could also lead to identify potential targets for the treatment of immune disorders by modulation of the ubiquitin pathway.

HYPOTHESIS

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Hypothesis:
The transcription factors PU.1 and E2A have important functions in T-cell development. Dysregulated transcription activation of either PU.1 or E2A alters T-cell development and causes immune disorders. The protein expression levels of both PU.1 and E2A are tightly controlled during the development of T cells. The up-regulation of these proteins is controlled at the transcriptional level. However, the mechanisms whereby these proteins are down-regulated at certain stages of T-cell development remain unknown. We hypothesize that ubiquitination, a post-translational modification that mediates protein degradation, eliminates transcription factors PU.1 and E2A to promote T-cell development. We propose to visualize PU.1 and E2A ubiquitination in the primary T cells at different developmental stages in mice.

Goals:
The goals of this proposed study are to determine the molecular mechanisms of ubiquitination in regulating the transcription activity of PU.1 and E2A in living cells, and the involvement of ubiquitination in T-cell development.

Methods:
The studies will take the advantages of an ubiquitin-mediated fluorescence complementation (UbFC) assay for imaging proteins modified by ubiquitin in living cells. This approach is based on the fluorescence complementation of two fragments of fluorescent proteins (FP) to ubiquitin and the target proteins. Once these fragments have been brought together by covalent modification, the intact fluorophore is reconstituted. The strong intrinsic fluorescence from complemented FP enables visualization of ubiquitination without the need to add extraneous fluorescent substrate and discontinue the cell culture. Therefore, this assay allows us to study when, how and where the covalent modification occurs in cells under conditions closely reflecting physiological situations.  In the study proposed we will use the UbFC assay to visualize PU.1 and E2A ubiquitinationto elucidate how ubiquitination regulates the subcellular localization, the protein stability, the nuclear-cytoplasm shuttling and the transcription activation of both PU.1 and E2A in living cells. We will visualize PU.1 and E2A ubiquitination during T-cell development in mice to determine at which T-cell developmental stage(s) PU.1 and E2A are ubiquitinated and how their ubiquitination is kinetically regulated during T-cell development.

SELECTED PUBLICATIONS

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Fang D. and Kerppola T.   Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/Aip4 is localized to endow-lysosome. Proc Natl Acad Sci U S A. 2004 Oct 12;101(41):14782-7.

Fang D., Elly C., Gao B., Fang N., Altman Y., Joazeiro C., Hunter T., Copeland N., Jenkins N., Liu Y.C.  Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation.  Nat Immunol. 2002 Mar;3(3):281-7.

Fang D. and Liu Y.C.  Proteolysis-independent regulation of phosphatidylinositol 3-kinase by Cbl-b-mediated ubiquitination in T cells.  Nat Immunol. 2001 Sep;2(9):870-5.