以原子尺度計算方法探討軟骨中蛋白聚糖降解的分子機制

Abstract

生物力學刺激在人體發育、成長與老化扮演相當重要的角色，正常的力學刺激可以增強人體骨骼的強度，然而不適當的力學刺激將會加速老化甚至引起許多相關疾病，以關節為例，髖關節發育不良是異常的力學刺激所造成的畸形關節發育病症，而軟骨受到過度的力學刺激甚至創傷時，會引發關節疼痛和功能障礙，引起退化性關節炎 (Osteoarthritis, OA) 等疾病。 這些疾病的產生，都與關節軟骨中異常的組織降解有關，其中Aggrecan被異常降解是這些疾病產生的主要原因之一。 正常狀態下，Aggrecan可以保護膠原蛋白不被降解，然而在異常的力學刺激下，Aggrecan的核心蛋白會被異常水解，導致第二型膠原蛋白失去Aggrecan的保護而進一步被水解，引起相關疾病，因此如欲完整了解這些疾病的成因，必須先了解Aggrecan降解的分子機制。 Aggrecan降解的重要區域為核心蛋白上位於G1和G2之間的區域，然而由於核心蛋白的分子結構尚未被完整解出，目前對於原子尺度下Aggrecan核心蛋白的降解機制仍不清楚。 本研究透過高速電腦所提供的計算能力，從原子尺度出發，以homology modeling的方法建立Aggrecan核心蛋白重要切割位點的分子模型，結合目前已知的蛋白水解酵素模型 (MMP-8)，利用原子尺度計算研究蛋白水解酵素與核心蛋白的結合機制，並進一步探討Aggrecan降解的分子機制。 我們發現，在catalytic cleavage site附近的兩個關鍵殘基：位於P2'帶正電荷的精氨酸和位於P3'帶有小側鏈的甘氨酸，它們在MMP-8和peptide之間形成穩定的binding pose，這種穩定的binding pose對於催化裂解位點的發生起到相當重要的作用。 在potential cleavage site附近的殘基沒有這些特徵，導致在potential cleavage site附近的MMP-8和peptide之間的binding pose不穩定。 從binding affinity來看，我們發現MMP-8在catalytic cleavage site的binding affinity比potential cleavage site更好。 穩定地與Aggrecan核心蛋白的catalytic cleavage site結合，使MMP-8保持“active”狀態，進而才能水解特定位置Aggrecan的核心蛋白。 相反，與Aggrecan核心蛋白potential cleavage site的不穩定的結合使MMP-8保持“inactive”狀態。 我們的研究在原子尺度上解釋了catalytic cleavage site附近的殘基如何使其被MMP-8裂解的機理。 這項研究的結果將在分子水平上增進我們對Aggrecan核心蛋白降解機理的理解。

Biomechanical forces play a critical role in our body. The musculoskeletal system generates, absorbs and transmits force, enabling the functional movement of our body. When trauma or excessive mechanical loading injures the articular cartilage, focal degradation of cartilage and remodeling of subchondral bone occur, resulting in joint pain and dysfunction, clinically identified as osteoarthritis (OA). These diseases are associated with abnormal degradation of the extracellular matrix in the articular cartilage. In the normal state, aggrecan can protect collagen from degradation. However, with the excessive mechanical loading, the core protein of aggrecan is hydrolyzed. It has been shown that the interglobular domain (IGD) of aggrecan core protein is highly sensitive to proteolysis. However, it is still not very clear about the degradation mechanism of aggrecan core protein at the molecular level. This study takes an innovative in silico approach to investigate the mechanism of aggrecan core protein degradation at the molecular level. We construct the full atomistic models of aggrecan core protein near the cleavage site. We study the specific enzyme (MMP-8) to understand the molecular mechanism of aggrecan core protein degradation. We found that the two key residues in the vicinity of catalytic site, arginine in P2’ who is in positively charged and glycine P3’ with a small side chain, which play an important role in forming a stable binding pose between MMP-8 and peptide near the catalytic cleavage site. Without these characteristics of residue in the vicinity of potential cleavage site, resulting in unstable binding pose between MMP-8 and peptide near the potential cleavage site. Seen from binging affinity, we find that it is more difficult for peptide near the catalytic cleavage site to unbind from MMP-8. But for peptide near the potential cleavage site, it is relatively easier to unbind, which is considered that MMP-8 has a better binding affinity at catalytic cleavage site than potential cleavage site. Stably binding to the catalytic cleavage site of aggrecan core protein make MMP-8 stay in an “active” state, and then hydrolyze the scissile bond of aggrecan core protein. On the contrary, unstably binding to the potential cleavage site of aggrecan core protein make MMP-8 stay in an “inactive” state. Our study explains the catalytic mechanism of how residues at the vicinity of the catalytic cleavage site enable it to be cleaved by MMP-8 at the atomistic scale. We anticipate that the results of this study will advance our understandings of the degradation mechanism in aggrecan core protein at the molecular level.