Roy J. Soberman, M.D.

Molecular Organization of Inflammation

Inflammation of islets is a major component of islet cell destruction in type 1 diabetes. The leukotrienes are inflammatory mediators that link innate and adaptive immunity. LTC₄ is a critical modulator of dendritic cell trafficking, and LTB₄ recruits and activates monocytes, macrophages and subsets of T-cells. Elucidating the mechanisms that control the synthesis and metabolism of leukotrienes are, therefore, critical in understanding the destruction of islet cells in Type 1 diabetes, and the regulation of host defenses in the peripheral vascular disease and associated infections seen in Type 2 diabetes.

The biosynthesis of leukotrienes (LTs) is under a complex set of controls. To form LTC₄ in response to cell activation at least 4 committed proteins must be localized to the outer nuclear membrane and ER (1-5). These are: cPLA₂, which releases arachidonic acid (AA) from membrane phospholipids, the 5-lipoxygenase (LO), the five-lipoxygenase-activating protein (FLAP) and leukotriene C₄ synthase (LTC₄ synthase). FLAP functions by presenting AA to the 5-LO, which then converts it to LTA₄. LTA₄ is then converted to LTC₄ by LTC₄ synthase or alternatively to LTB₄ by LTA₄ hydrolase. FLAP and LTC₄synthase are closely related 17 kDa integral membrane proteins. Until recently, how, or even whether, these molecules interacted was unknown (1-5).

Our laboratory, using a combination of live-cell fluorescent lifetime imaging (FLIM), supported by biochemical analysis with in situ cross-linking, and co-immunoprecipitation, has identified the presence of a heterotrimeric complex of LTC₄ synthase and FLAP that is present in resting leukocytes and organizes the synthesis of leukotrienes. This is likely to be a heterotrimeric enzyme with a single active site composed of domains from two molecules of LTC₄ synthase and one molecule of FLAP, allowing the direct conversion of AA to LTC₄. Presumably, 5-LO is incorporated into this complex, but we have not completed the final analysis of its interactions. The close structural identity of FLAP and LTC₄ synthase are critical in regulating the interaction between these two proteins. We are using a combination of live-cell FLIM combined with biochemical approaches and Mass Spectrometry to achieve three goals: 1. Map the interacting domains of FLAP and LTC₄ synthase. 2. Identify novel interacting proteins that are recruited to this complex during cell activation, including 5-LO and LTA₄ hydrolase. 3. Develop in vivo models of inflammation to monitor the interactions and assembly of the leukotriene biosynthetic complex using in vivo FLIM.

The organization of the nuclear response to oxidative stress. Cytochrome P450 4F3 down-regulates inflammation by inactivating LTB₄ (6-8). To understand the regulation of CYP4F3 expression, we examined its distribution in maturing populations of human bone marrow cells and identified the splicing pathways. CYP4F3A is expressed in myeloid cells; its expression is coordinate with known myeloid differentiation markers such as CD11b, and also increases concomitantly with myeloperoxidase during development. In contrast, CYP4F3B is expressed in lymphocytes, and expression is restricted to a small population (~10%) of CD3+ T cells. We determined that alternative promoters regulate lineage-specific expression of CYP4F3 isoforms in myeloid cells, lymphoid cells, and liver. Surprisingly, activity of the myeloid-specific promoter could not be accounted for by known myeloid transcription factors including PU.1 and MZF-1, but it could be activated by ZEB-2 and CtBP. The results suggest new roles for these proteins in myeloid transcription. CtBP is a sensor of nuclear NAD+ and nuclear redox tone. In addition, the expression of CYP4F3 and its murine orthologue CYP4F18 are up-regulated 20-30 fold during the maturation of dendritic cells, a processes that allows these cells to present antigen with maximal efficiency. The induction of CYP4F18 is redox sensitive, and abolished by the interruption of redox signaling. CYP4F18 induction involves additional and distinct redox-regulated transcription processes independent of CtBP that are controlled by the protein nucleoredoxin. Our laboratory is using a combination of genetic and proteomic approaches to identify proteins that interact with CtBP and nucleoredoxin. We are also identifying the redox-sensitive interactions of nucleoredoxin in dendritic cell nuclei with the goal of understanding the organization of redox signaling and the nuclear response to oxidative stress. A final goal of this series of experiments is to understand a likely role of nuclear redox tone in regulating dendritic cell maturation.

References:

1. Christmas, P., Fox, J.W., Ursino, S.R., and Soberman, R.J. Differential Localization of 5- and 15-Lipoxygenases to the Nuclear Envelope in RAW Macrophages. J. Biol. Chem., 1999; 274: 25594 - 25598.

2. Christmas, P. Weber, B.M., McKee, M., Brown, D., and Soberman, R.J. Membrane Localization and Topology of Leukotriene C₄ Synthase. J. Biol. Chem.,2002; 277: 28902 - 28908.

3. Soberman, R.J., and Christmas, P. The Organization and Consequences of Eicosanoid Signaling. J. Clin. Invest., 2003; 111:1107-1113.

4. Soberman, R.J. Series Introduction: The Expanding Network of Redox Signaling: New Observations, Complexities, and Perspectives. J. Clin. Invest., 2003; 111:571-574.

5. Fitzpatrick, F.A., and Soberman, R.J., Regulated Formation of Eicosanoids. J. Clin. Invest., 2001; 107:1347-1351.

6. Christmas, P., Jones, J.P., Patten, C.J., Rock, D.A., Zheng, Y., Cheng, S-M., Weber, B.M., Carlesso, N., Scadden, D.T., Rettie, A.E., and Soberman, R.J. Alternative Splicing Determines the Function of CYP4F3 by Switching Substrate Specificity. J. Biol. Chem., 2001; 276: 38166 - 38172.

7. Christmas, P. Ursino, S., Fox, J.W., and Soberman, R.J. Expression of the CYP4F3 Gene. tissue-specific splicing and alternative promoters generate high and low k_m forms of leukotriene b₄ -hydroxylase. J. Biol. Chem., 1999; 274: 21191 - 21199.

8. Christmas, P., Carlesso, N., Shang, H., Cheng, S-M., Weber, B.M., Preffer, F.I., Scadden, D.T., and Soberman, R.J. Myeloid Expression of Cytochrome P450 4F3 is Determined by a Lineage-Specific AlternativePromoter. J Biol Chem. 2003;278:25133-42.

9. Mandal AK, Skoch J, Bacskai BJ, Hyman BT, Christmas P, Miller D, Yamin TT, Xu S, Wisniewski D, Evans JF, Soberman RJ. The membrane organization of leukotriene synthesis.Proc Natl Acad Sci U S A. 2004;101:6587-92.


	Roy J. Soberman, M.D. Molecular Organization of Inflammation Inflammation of islets is a major component of islet cell destruction in type 1 diabetes. The leukotrienes are inflammatory mediators that link innate and adaptive immunity. LTC₄ is a critical modulator of dendritic cell trafficking, and LTB₄ recruits and activates monocytes, macrophages and subsets of T-cells. Elucidating the mechanisms that control the synthesis and metabolism of leukotrienes are, therefore, critical in understanding the destruction of islet cells in Type 1 diabetes, and the regulation of host defenses in the peripheral vascular disease and associated infections seen in Type 2 diabetes. The biosynthesis of leukotrienes (LTs) is under a complex set of controls. To form LTC₄ in response to cell activation at least 4 committed proteins must be localized to the outer nuclear membrane and ER (1-5). These are: cPLA₂, which releases arachidonic acid (AA) from membrane phospholipids, the 5-lipoxygenase (LO), the five-lipoxygenase-activating protein (FLAP) and leukotriene C₄ synthase (LTC₄ synthase). FLAP functions by presenting AA to the 5-LO, which then converts it to LTA₄. LTA₄ is then converted to LTC₄ by LTC₄ synthase or alternatively to LTB₄ by LTA₄ hydrolase. FLAP and LTC₄synthase are closely related 17 kDa integral membrane proteins. Until recently, how, or even whether, these molecules interacted was unknown (1-5). Our laboratory, using a combination of live-cell fluorescent lifetime imaging (FLIM), supported by biochemical analysis with in situ cross-linking, and co-immunoprecipitation, has identified the presence of a heterotrimeric complex of LTC₄ synthase and FLAP that is present in resting leukocytes and organizes the synthesis of leukotrienes. This is likely to be a heterotrimeric enzyme with a single active site composed of domains from two molecules of LTC₄ synthase and one molecule of FLAP, allowing the direct conversion of AA to LTC₄. Presumably, 5-LO is incorporated into this complex, but we have not completed the final analysis of its interactions. The close structural identity of FLAP and LTC₄ synthase are critical in regulating the interaction between these two proteins. We are using a combination of live-cell FLIM combined with biochemical approaches and Mass Spectrometry to achieve three goals: 1. Map the interacting domains of FLAP and LTC₄ synthase. 2. Identify novel interacting proteins that are recruited to this complex during cell activation, including 5-LO and LTA₄ hydrolase. 3. Develop in vivo models of inflammation to monitor the interactions and assembly of the leukotriene biosynthetic complex using in vivo FLIM. The organization of the nuclear response to oxidative stress. Cytochrome P450 4F3 down-regulates inflammation by inactivating LTB₄ (6-8). To understand the regulation of CYP4F3 expression, we examined its distribution in maturing populations of human bone marrow cells and identified the splicing pathways. CYP4F3A is expressed in myeloid cells; its expression is coordinate with known myeloid differentiation markers such as CD11b, and also increases concomitantly with myeloperoxidase during development. In contrast, CYP4F3B is expressed in lymphocytes, and expression is restricted to a small population (~10%) of CD3+ T cells. We determined that alternative promoters regulate lineage-specific expression of CYP4F3 isoforms in myeloid cells, lymphoid cells, and liver. Surprisingly, activity of the myeloid-specific promoter could not be accounted for by known myeloid transcription factors including PU.1 and MZF-1, but it could be activated by ZEB-2 and CtBP. The results suggest new roles for these proteins in myeloid transcription. CtBP is a sensor of nuclear NAD+ and nuclear redox tone. In addition, the expression of CYP4F3 and its murine orthologue CYP4F18 are up-regulated 20-30 fold during the maturation of dendritic cells, a processes that allows these cells to present antigen with maximal efficiency. The induction of CYP4F18 is redox sensitive, and abolished by the interruption of redox signaling. CYP4F18 induction involves additional and distinct redox-regulated transcription processes independent of CtBP that are controlled by the protein nucleoredoxin. Our laboratory is using a combination of genetic and proteomic approaches to identify proteins that interact with CtBP and nucleoredoxin. We are also identifying the redox-sensitive interactions of nucleoredoxin in dendritic cell nuclei with the goal of understanding the organization of redox signaling and the nuclear response to oxidative stress. A final goal of this series of experiments is to understand a likely role of nuclear redox tone in regulating dendritic cell maturation. References: 1. Christmas, P., Fox, J.W., Ursino, S.R., and Soberman, R.J. Differential Localization of 5- and 15-Lipoxygenases to the Nuclear Envelope in RAW Macrophages. J. Biol. Chem., 1999; 274: 25594 - 25598. 2. Christmas, P. Weber, B.M., McKee, M., Brown, D., and Soberman, R.J. Membrane Localization and Topology of Leukotriene C₄ Synthase. J. Biol. Chem.,2002; 277: 28902 - 28908. 3. Soberman, R.J., and Christmas, P. The Organization and Consequences of Eicosanoid Signaling. J. Clin. Invest., 2003; 111:1107-1113. 4. Soberman, R.J. Series Introduction: The Expanding Network of Redox Signaling: New Observations, Complexities, and Perspectives. J. Clin. Invest., 2003; 111:571-574. 5. Fitzpatrick, F.A., and Soberman, R.J., Regulated Formation of Eicosanoids. J. Clin. Invest., 2001; 107:1347-1351. 6. Christmas, P., Jones, J.P., Patten, C.J., Rock, D.A., Zheng, Y., Cheng, S-M., Weber, B.M., Carlesso, N., Scadden, D.T., Rettie, A.E., and Soberman, R.J. Alternative Splicing Determines the Function of CYP4F3 by Switching Substrate Specificity. J. Biol. Chem., 2001; 276: 38166 - 38172. 7. Christmas, P. Ursino, S., Fox, J.W., and Soberman, R.J. Expression of the CYP4F3 Gene. tissue-specific splicing and alternative promoters generate high and low k_m forms of leukotriene b₄ -hydroxylase. J. Biol. Chem., 1999; 274: 21191 - 21199. 8. Christmas, P., Carlesso, N., Shang, H., Cheng, S-M., Weber, B.M., Preffer, F.I., Scadden, D.T., and Soberman, R.J. Myeloid Expression of Cytochrome P450 4F3 is Determined by a Lineage-Specific AlternativePromoter. J Biol Chem. 2003;278:25133-42. 9. Mandal AK, Skoch J, Bacskai BJ, Hyman BT, Christmas P, Miller D, Yamin TT, Xu S, Wisniewski D, Evans JF, Soberman RJ. The membrane organization of leukotriene synthesis.Proc Natl Acad Sci U S A. 2004;101:6587-92.