We plan to continue work in four closely related areas: (1) chitin synthesis in yeast; (2) chitin synthesis and glycosylation in multicellular organisms: (3) the enzymology and biology of the hyaluronan/chitin oligosaccharide synthesizing enzyme DG42, and (4) properties of chitin deacetylases and """"""""antifungal"""""""" chitinases. Yeast chitin synthase 3 (Chs3p) is targeted to the bud-neck region of the cell where it synthesizes the chitin ring. It also functions throughout the plasma membrane to """"""""reinforce"""""""" the cell wall when it has been weakened. Our immediate goals will be to compare targeting and activation of Chs3p when it is engaged either in building the bud-neck structure or depositing chitin in the lateral wall. In insects, chitin is present in the cuticle and in the intestinal peritrophic membrane. Insect genomes examined to date have two chitin synthase genes, while most filamentous fungi have at least six. We will study the enzymology of individual proteins that have been expressed in appropriate heterologous systems. We will also investigate when and where the genes are transcribed and where within cells the enzymes are localized. From our work as well as work in other laboratories, it is clear that some hyaluronan synthases (HAS enzymes, including DG42) are able to synthesize either hyaluronan (a polymer of alternating glucuronic acid and acetylglucosamine residues) or chitin oligosaccharide (containing only acetylglucosamine) depending on conformation of the enzyme and/or incubation conditions. In other developments, the Spaink group have shown clearly that the chitin tetrasaccharide, and only the tetrasaccharide, is able to restore development of the anterior-posterior axis in zebrafish inhibited with DG42 antisense RNA. We will continue our own enzymatic work and will initiate collaborative X-ray crystallographic and biological studies. In our fourth project, we will focus on chitin deacetylases, apparently soluble proteins that must act on nascent chitin chains before they associate into fibers. and on """"""""antifungal"""""""" chitinases as potential drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047485-14
Application #
6862739
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
1992-05-01
Project End
2007-02-28
Budget Start
2005-03-01
Budget End
2007-02-28
Support Year
14
Fiscal Year
2005
Total Cost
$305,775
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Van Horn, Wade D; Beel, Andrew J; Kang, Congbao et al. (2010) The impact of window functions on NMR-based paramagnetic relaxation enhancement measurements in membrane proteins. Biochim Biophys Acta 1798:140-9
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Van Horn, Wade D; Kim, Hak-Jun; Ellis, Charles D et al. (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324:1726-9

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