Rodríguez-Moreno María, Legaz Isabel
Department of Legal and Forensic Medicine, Biomedical Research Institute of Murcia (IMIB), Regional Campus of International Excellence "Campus Mare Nostrum", Faculty of Medicine, University of Murcia (UMU), 30100 Murcia, Spain.
Biomedicines. 2025 Feb 21;13(3):544. doi: 10.3390/biomedicines13030544.
Chronic wounds, such as diabetic ulcers, often fail to progress through healing due to persistent inflammation, infections, and extracellular matrix (ECM) imbalances. Cathepsin D, an aspartate protease active in acidic environments, plays a pivotal role in wound healing by mediating inflammatory responses, ECM remodeling, and macrophage phenotype transitions. Its dysregulation, however, can impair healing, highlighting the need for targeted modulation of its activity. The aim of this study was to investigate the molecular interaction between Fe and cathepsin D's catalytic core and ionic zipper under physiological and acidic conditions to identify strategies to enhance tissue repair and accelerate the healing of chronic wounds. The molecular structure of active cathepsin D was obtained from the Protein Data Bank (PDB) and analyzed using UCSF Chimera. Molecular interactions between cathepsin D and ferrous ions (Fe) were studied, focusing on key residues (D33 and D231) and ionic zipper residues (E5, E180, and D187). Our results showed that the active form of cathepsin D, a 96 kDa dimer, consisted of heterodimers with distinct amino acid chains, where residues D33 and D231 formed the active site, and E5, E180, and D187 constituted the ionic zipper. A functional pocket containing the conserved residues D33 and D231, essential for proteolytic activity, was identified. At physiological pH (7.5), D33 exhibited the most potent interactions with Fe, with interaction energies of -7 × 10 J at oxygen atoms of the carboxylate group (OD1) and α-carbon (CA) atoms, whereas D231 showed slightly lower energies of -6 × 10 J at γ-carbon atom (CG) and CA atoms. At acidic pH (4), E5 was the primary interacting residue, with the shortest distance to Fe (2.69 Å), and showed stable interactions across several atoms, emphasizing its role in metal binding. pH conditions strongly influence the interaction of cathepsin D with Fe. At physiological pH, residues D33 and D231 demonstrate robust and energetically efficient binding with Fe. At the same time, under acidic conditions, E5 emerges as the primary residue involved, potentially affecting the ionic zipper of cathepsin D. These insights provide a molecular foundation for targeting specific residues to modulate cathepsin D activity, presenting promising opportunities for therapeutic strategies aimed at improving chronic wound healing.
慢性伤口，如糖尿病溃疡，由于持续的炎症、感染和细胞外基质（ECM）失衡，常常无法顺利愈合。组织蛋白酶D是一种在酸性环境中具有活性的天冬氨酸蛋白酶，通过介导炎症反应、ECM重塑和巨噬细胞表型转变，在伤口愈合中起关键作用。然而，其失调会损害愈合，这凸显了对其活性进行靶向调节的必要性。本研究的目的是研究在生理和酸性条件下铁与组织蛋白酶D的催化核心和离子拉链之间的分子相互作用，以确定增强组织修复和加速慢性伤口愈合的策略。活性组织蛋白酶D的分子结构从蛋白质数据库（PDB）中获取，并使用UCSF Chimera进行分析。研究了组织蛋白酶D与亚铁离子（Fe）之间的分子相互作用，重点关注关键残基（D33和D231）和离子拉链残基（E5、E180和D187）。我们的结果表明，组织蛋白酶D的活性形式为96 kDa二聚体，由具有不同氨基酸链的异二聚体组成，其中残基D33和D231形成活性位点，E5、E180和D187构成离子拉链。鉴定出一个包含保守残基D33和D231的功能性口袋，这对蛋白水解活性至关重要。在生理pH（约7.5）下，D对铁的相互作用最为强烈，在羧基（OD1）的氧原子和α-碳原子（CA）处的相互作用能为-7×10 J，而D231在γ-碳原子（CG）和CA原子处的能量略低，为-6×10 J。在酸性pH（约4）下，E5是主要的相互作用残基，与铁的距离最短（2.69 Å），并且在多个原子上表现出稳定的相互作用，强调了其在金属结合中的作用。pH条件强烈影响组织蛋白酶D与铁的相互作用。在生理pH下，残基D33和D231与铁表现出强大且能量高效的结合。同时，在酸性条件下，E5成为主要涉及的残基，可能影响组织蛋白酶D的离子拉链。这些见解为靶向特定残基调节组织蛋白酶D活性提供了分子基础，为旨在改善慢性伤口愈合的治疗策略带来了有希望的机会。
