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Proteolytic enzymes in skin physiology and pathophysiology

Grant number: 14/18877-9
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): March 01, 2015
Effective date (End): February 28, 2017
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal Investigator:Maria Aparecida Juliano
Grantee:Carolina Bellini Parise
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Associated research grant:12/50191-4 - Synthesis, kinetic studies and applications of substrates and inhibitors for proteolytic enzymes, AP.TEM

Abstract

Skin is the first barrier protection against injuries, infections and is involved in immunoregulatory mechanisms. Proteases are play roles in these physiological and pathophysiological functions including skin desquamation. This project will focus on two tissue kallikrein 5 and7 (KLK5 and KLK7) highly expressed and functional in human skin. Both are serine proteases, but KLK5 such as trypsin hydrolyzes its substrate in arginine (R) and KLK7 in tyrosine (Y) or phenylalanine (F) similar to chymotrypsin. However the specificities of these proteases are not restricted to the site of hydrolysis but depend on neighboring amino acids, and are activated or inhibited by components of the skin as described recently by Dr. Maria A. Juliano and her group with KLK76. This study showed for the first time that a kallikrein may have semaphorin as substrate, in particular Sema 6-B. Semaphorins are axon growth guides molecules with attraction or repulsion activities on the nerve cells and endothelial cells. Preliminary modeling of KLK7 with the peptide derived from Sema 6-B unexpected interactions were observed and suggested possible roles of the areas with positive charges on the surface of KLKs 5 and 7 to bind the substrates. In view of these observations, we will complete studies on the specificity of these two proteases, we will map the electrostatic interactions with peptides containing negative load sequences. Furthermore, with the sequences of Semas 3-A (present in the skin), 6-B (in glioblastomas) and nerve growth factor (in skin) and we will synthesize peptides with 11 amino acids placing the center of each R, Y or F. We selected 158 sequences that will be synthesized as FRET peptides (Fluorescence Resonance Energy Transfer) type Abz-peptide-EDDnp with the fluorescence pair as donor (Abz) / acceptor (EDDnp) and tested as substrates or inhibitors for KLK5 the peptides with R and with KLK7 the peptides with Y or F. The action of KLKs 5 and 7 are discussed in the whole protein Sema 3-A and 6-B that are commercially available. Obtaining inhibitors for KLKs 5 and 7 will be on the agenda in this work. These will be synthesized as FRET peptides (Fluorescence Resonance Energy Transfer) type Abz-peptide-EDDnp with the pair donor (Abz) / acceptor (EDDnp) fluorescence and tested as substrates or inhibitors for KLK5 peptides with R and KLK7 peptides with Y or F. The action of KLKs 5 and 7 shall be examined at whole protein Sema 3-A and 6-B that are commercially available. Obtaining inhibitors for KLKs 5 and 7 will be on the agenda in this work. Previous studies by Dr. Carolina shown that human myelomonocytic cell line THP-1 can present a profile of antigen presenting cells (APC) towards sensitizing chemicals. From these data, we propose the proteolytic profile analysis using FRET and fluorogenic peptides in THP-1 cells to identify effects of chemical sensitizers compounds and natural protease inhibitors, mimicking what may occur in the skin. (AU)