REPOSITORY > RESULTS

Abstract

Proteases are responsible for enzymatic cleavage of peptide bonds, which is a basic requirement for
completion of diverse biological processes.The proteolytic activity of proteases must be rigorously
controlled to avoid inappropriate degradation of proteins. Imbalance in regulation of proteolytic activity
can be found in a wide range of diseases. Degradation of articular cartilage and bone is a characteristic
feature of various types of arthritic diseases, including rheumatoid arthritis (RA) and osteoarthritis (OA).
These irreversible changes are probably mediated by the excessive protease activity resulting from
increased expression of matrix metalloproteinase (MMPs) and cysteine cathepsins families and/or an
altered equilibrium between these proteases and their endogenous inhibitors. In addition, proinflammatory
cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), are
believed to contribute to tissue damage in arthritis by inducing the release of tissue-degrading proteases. A
detailed comparison between the protease levels and activities between RA samples and OA samples has
never been made. The aim of our study was to determine the protein levels of certain proinflammatory
cytokines (IL-1β, IL-6 and TNF-α), selected cysteine cathepsins (cathepsin B and S) and matrix
metalloproteases (MMP-1, -3 and -13) as well as their proteolytic activities in samples of synovial fluid
from patients with either RA or OA. We showed that both cysteine cathepsins B and S and matrix
metalloproteases-1, -3 and -13 have been detected in patient synovial fluid samples with significantly
higher levels detected in rheumatoid arthritis patients. Among the proteases, cathepsin S was found to be
significantly elevated, consistent with its critical role in the immune response. These results suggest that
cysteine cathepsins have a major role in inflammation at least in RA. In addition to proteases, interleukin-6
was detected at significant levels in most samples, suggesting that proinflammatory cytokines may be
involved in the stimulation of expression of these proteases during inflammation.
Citrullination is a posttranslational modification in a protein where arginine is converted into the non-
natural amino acid citrulline. This process is catalyzed by the enzyme peptidylarginine deiminase (PAD). It
is believed that citrullination can convert a nonimmunogenic protein into an autoimmunogenic protein.
Consistent with this idea, citrullinated proteins as well as antibodies against citrullinated proteins are found
in patients with RA. Moreover, antibodies against citrullinated proteins have become a reliable diagnostic
marker for RA. In addition, citrullination may affect protein degradation as molecular interactions that are
mediated by positively charged arginine residues are lost upon citrullination. The goal of this study was to
evaluate the effect of citrullination of extracellular matrix proteins on their susceptibility for degradation by
proteases that have been implicated into articular cartilage and bone erosion in RA. Since type I and type II
collagens are major components of articular cartilage and bone matrix, they also represent our model
proteins for this study. We managed to perform in vitro citrullination of gelatine type I with a commercially
available PAD enzyme at 37 °C. Successful citrullination was confirmed using antibodies against
citrullinated proteins. We have showed that citrullinated form of geltine type I is more susceptible for
degradation by cysteine cathepsin B, K and S than non-citrullinated. These results were not confirmed
using MMP-1 and MMP-3. On the other hand we were not able to perform citrullination of native form of
type II collagen under these experimental conditions.