Characterization of human skin fibroblasts elastase activity

Area of Science:

• Biochemistry
• Dermatology
• Enzymology

Background:

• Human Skin Fibroblasts (HSF) play a crucial role in skin structure and integrity.
• Understanding the enzymatic activities within HSF is vital for skin biology and aging research.

Purpose of the Study:

• To characterize the enzyme(s) responsible for hydrolyzing Succinoyl trialanine paranitroanilide (Suc(Ala)3NA) in HSF.
• To investigate the role of these enzymes in the degradation of human skin elastic fibers.

Main Methods:

• Enzyme activity assays using Suc(Ala)3NA substrate.
• Inhibition studies with metal chelators, Copper, Zinc, and Tissue Inhibitor of Metallo Proteases (TIMP).
• Chromatographic separation (ion exchange and HPLC) to isolate enzyme activities.
• Elastinolytic activity assessment on insoluble elastin and human skin sections.
• Automated image analysis for quantitation of fiber degradation.

Main Results:

• HSF enzyme activity involves both endopeptidase and aminopeptidase(s).
• Activities were inhibited by metal chelators and ions (Cu2+, Zn2+), but not TIMP.
• A specific endopeptidase, HSF E1 (Mr 94,000), was identified and purified.
• HSF E1 extensively degrades human skin elastic fibers, with activity increasing with fibroblast passage.

Conclusions:

• The primary enzyme responsible for Suc(Ala)3NA hydrolysis in HSF is an endopeptidase (HSF E1) acting in concert with aminopeptidases.
• HSF E1 is a key enzyme in the degradation of dermal elastic fibers.
• Fibroblast passage number influences the level of HSF E1, suggesting a role in skin aging.