- This is an amended competitive renewal application. The broad objective of the proposed research is to determine the biochemical structures, biophysical interactions, and biological functions of epidermal lipids. The results of the studies described are expected to advance the understanding of the epidermal barrier function of the stratum corneum. The first specific aim is to determine the manner in which omega-hydroxyceramides are attached to protein on the exterior of the corneocyte protein envelope in pig and human stratum corneum. The hypothesis being tested is that all of the hydroxyceramide molecules are bound to the protein envelope through their omega-hydroxyl function while, in addition, half of the sphingosine head groups are derivatized in some other way. Lipids bound to corneocytes will be analyzed by isopropylidene protection of those sphingosine head groups that have both hydroxyl groups free, followed by chromic acid oxidation of free hydroxyl groups, and subsequent separation and NMR structure determination. The second specific aim is to determine the molecular conformation of involucrin that enables this corneocyte envelope surface protein to support the organized lipid envelope. Peptides representing specific sequences of human involucrin will be synthesized and the molecular conformations of these peptides, with and without model lipids attached, will be determined by circular dichroism and NMR. The third specific aim is to determine how ceramides of the long-chain omega-hydroxyacids control the arrangement of the intercellular lamellae in the stratum corneum. The effects of natural and synthetic derivatives of 30-carbon omega-hydroxyacids on the biophysical properties of the lipid lamellae will be assessed by X-ray diffraction and electron microscopy. The fourth specific aim addresses the steps in the formation of lamellar disks from lipid vesicles and of paired lamellar sheets from the lamellar disks. Studies in vitro are designed to determine factors influencing these processes. The composition of liposomes formed from stratum corneum lipids will be varied so that bulky and/or charged and polar groups are on the exterior; agents expected to enhance flattening and stacking will be evaluated. The resulting forms will be assessed by electron microscopy.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Moshell, Alan N
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University of Iowa
Schools of Medicine
Iowa City
United States
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Stewart, M E; Downing, D T (2001) The omega-hydroxyceramides of pig epidermis are attached to corneocytes solely through omega-hydroxyl groups. J Lipid Res 42:1105-10
Lazo, N D; Downing, D T (2001) Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices. J Pept Res 58:457-63
Stewart, M E; Downing, D T (1999) A new 6-hydroxy-4-sphingenine-containing ceramide in human skin. J Lipid Res 40:1434-9
Lazo, N D; Downing, D T (1999) A mixture of alpha-helical and 3(10)-helical conformations for involucrin in the human epidermal corneocyte envelope provides a scaffold for the attachment of both lipids and proteins. J Biol Chem 274:37340-4
Lazo, N D; Downing, D T (1999) Fibril formation by amyloid-beta proteins may involve beta-helical protofibrils. J Pept Res 53:633-40
Lazo, N D; Downing, D T (1998) Amyloid fibrils may be assembled from beta-helical protofibrils. Biochemistry 37:1731-5
Lazo, N D; Downing, D T (1998) Stabilization of amphipathic alpha-helical and beta-helical conformations in synthetic peptides in the presence and absence of ionic interactions. J Pept Res 51:85-9
Lazo, N D; Downing, D T (1997) Circular dichroism of model peptides emulating the amphipathic alpha-helical regions of intermediate filaments. Biochemistry 36:2559-65
Lazo, N D; Downing, D T (1997) Beta-helical fibrils from a model peptide. Biochem Biophys Res Commun 235:675-9
Downing, D T (1996) Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations. Proteins 25:215-24

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