Our laboratory uses the fruit fly, Drosophila melanogaster, as a model system to understand the mechanisms by which animal cells sense the requirements for (and regulate the production of) fatty acids. As known from mammals, a complicated assemblage of protein machinery, known as the SREBP pathway, is central to these processes.
The SREBPs (sterol regulatory element binding proteins) are membrane-bound transcription factors. When lipid supplies are sufficient, they remain in the membranes of the endoplasmic reticulum - when cellular demand for lipid rises, another protein, SCAP, escorts SREBP to the Golgi apparatus where two different proteases (the site-1 protease (S1P), the site-2 protease (S2P)) are required to release the transcription factor from the membrane and enable it to travel to the nucleus. There, SREBP binds to DNA sequences in the upstream region of target genes and mediates their increased transcription.
The best studied role of the SREBP pathway in mammals is the regulation of cholesterol uptake and production but the SREBPs are also involved in aspects of fatty acid metabolism as well. Their role in fatty acid metabolism is easier to study in flies than in mammals. Vertebrates are capable of making their own cholesterol as well as their own fatty acids and have two separate genes that encode three different SREBP proteins. The multiplicity of inputs (sterol and fatty acid levels) and outputs (transcription of genes of sterol synthesis and uptake by SREBP-2 vs. transcription of genes of fatty acid synthesis by SREBP-1a and -1c) makes teasing apart the regulatory networks controlling aspects of fatty acid metabolism more complicated in mammals than in flies. Flies cannot make cholesterol from scratch but must get it as part of their diet. Their genome encodes only a single SREBP gene rather than the two in mammals. In addition, the considerable array of genetic and molecular tools available for Drosophila make it a productive experimental system in which to study aspects of fatty acid metabolism that are common to all animals.
In earlier work, we demonstrated that the SREBP pathway in Drosophila cells responds to palmitate rather than sterols. In dissecting the mechanisms responsible for palmitate regulation of the SREBP pathway, we discovered a metabolic cycle in which palmitate serves as a catalyst in the conversion of serine and ATP into phosphoethanolamine, which is the head group of phosphatidylethanolamine (PE). We went on to demonstrate that PE rather than palmitate itself, is the principle regulatory lipid in flies. Our extensive characterization of the SREBP pathway in Drosophila cells enabled us to use that system to show that mammalian Insig proteins were both necessary and sufficient to confer sterol regulation on the mammalian SREBP pathway. This was possible because flies have no Insig-like proteins, and this lack correlates with the absence of any sterol regulation of the SREBP pathway in flies.
Continuing to use mammalian cell genetics, we and colleagues identified two distinct roles for cholesterol in mammalian cells: 1) a bulk requirement that could be satisfied by several different sterols, and which was not enantioselective, and 2) a specific requirement for very small amounts of cholesterol itself.
We isolated mutant flies lacking the gene for SREBP and discovered that these animals die at the 2nd-to-3rd larval instar transition. We overcame this lethality by supplementing the larval diet with extra lipid. Free fatty acids were far more effective at promoting survival than were more complex lipids (e.g. triglycerides and phospholipids.) Our ability to recover adult flies lacking all SREBP function allowed us to show, in collaboration with Sara Cherry and Norbert Perrimon, that SREBP is essential for the replication of certain kinds of polio-like viruses.
Mammalian cells cannot survive if they lack components of the SREBP processing machinery such as, S1P, S2P, or Scap. Recently, we showed that flies lacking the site-2 protease (S2P) are, by contrast, viable. In Drosophila larvae, in the absence of S2P, another protease, the caspase Drice, cleaves SREBP, freeing it from the membrane, and enabling it to travel to the nucleus to mediate the increased transcription of target genes. This transcription is sufficient to enable flies to survive in the absence of S2P and cleavage of SREBP is one of a growing list of non-apoptotic functions of caspases.
Currently, work in the lab is aimed at finding the fly protein that functions in fatty acid regulation analogously to the Insig proteins in vertebrate sterol metabolism. We are studying the principal SREBP target genes in flies by biochemical and genetic analysis in order to understand the mechanism through which the activity of these genes feeds back onto the global regulation of lipid metabolism via the SREBP pathway. We are also conducting genome-wide screens for modifiers of SREBP activity in Drosophila to identify additional targets of SREBP transcriptional up-regulation.
RESEARCH INTERESTS
Genetics of the control of lipid metabolism in insects
Mammalian and insect somatic cell genetics
Evolution of the SREBP pathway
Cell biology of Site-2 protease
RECENT PUBLICATIONS
Amarneh, B., Matthews, K.A., and Rawson, R.B., "Activation of SREBP by the caspase drice in Drosophila larvae." J. Biol. Chem, v. 284, no. 15:9674-9682, April 2009
Matthews, K. M., Kunte, A. S., Tambe-Ebot, E., and Rawson, R. B., "Alternative Processing of SREBP during Larval Development in Drosophila melanogaster" Genetics, 81:119-128, January 2009
Kunte, A. S., Matthews, K. M., and Rawson, R. B., "Fatty acid auxotrophy in Drosophila larvae lacking SREBP" Cell Metabolism, 3:439-448, June 2006
Xu F., Rychnovsky, S.D., Belani, J.D., Hobbs H.H., Cohen J.C., Rawson R.B., "Dual roles for cholesterol in mammalian cells" PNAS (USA), 102:14551-14556, October 2005
SIGNIFICANT PUBLICATIONS
Kunte, A. S., Matthews, K. M., and Rawson, R. B., "Fatty acid auxotrophy in Drosophila larvae lacking SREBP" Cell Metabolism, 3:439-448, June 2006
Dobrosotskaya, I. Y., Seegmiller, A. C., Brown, M. S., Goldstein, J. L., and Rawson, R. B., "Regulation of SREBP Processing and Membrane Lipid Production by Phospholipids in Drosophila." Science, 269:879-883, 2002
Amarneh, B., Matthews, K.A., and Rawson, R.B., "Activation of SREBP by the caspase drice in Drosophila larvae." J. Biol Chem., e pub ahead of print, February 2009
Seegmiller, A. C., Dobrosotskaya, I., Goldstein, J. L., Ho, Y. K., Brown, M. S., Rawson, R.B., "The SREBP Pathway in Drosophila: Regulation by Palmitate, Not Sterols." Developmental Cell, 2:229-238, 2002
Rawson, R. B., Zelenski, N. G., Nijhawan, D., Ye, J., Sakai, J., Hasan, M. T., Chang, T. Y., Brown, M. S., and Goldstein, J. L., "Complementation Cloning of S2P, a Gene Encoding a Putative Metalloprotease Required for Intramembrane Cleavage of SREBPs." Molecular Cell, 1:47-57, 1997
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