Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.

细胞外蛋白水解和周转是组织稳态的核心过程。主要的基质降解酶是基质金属蛋白酶 (MMP) 家族的成员。除了处理细胞外、质膜和细胞内区室中的一系列其他因子外，MMP 还广泛降解核心基质成分。MMP 的蛋白水解活性由金属蛋白酶组织抑制剂 (TIMP) 调节，TIMP 是四个多功能基质体蛋白的家族，具有广泛的 MMP 抑制功能。因此，MMP 活性和 TIMP 水平之间良好调节的平衡被认为对于健康组织稳态至关重要，并且这种平衡可能在病理过程中长期受到干扰。MMP 和 TIMP 之间的关系很复杂，并且由于各种 MMP（特别是明胶酶）和 TIMP 家族成员之间的二次相互作用而缺乏典型酶抑制剂关系的约束。我们通过描述 MMP9 在摩尔过量时如何切割 TIMP 家族的成员来说明该系统中的新复杂性。TIMP 的蛋白水解加工可以产生具有潜在新属性的功能改变的肽。我们在此证明所有 TIMP 的 C 末端尾部均被摩尔过量的 MMP9 裂解。该处理去除了 TIMP3 的 N-糖基化位点，并防止 TIMP2 与潜在的 proMMP2 相互作用，这是细胞表面 MMP14 介导的 proMMP2 激活的先决条件。TIMP2/4 进一步裂解，产生~14 kDa N 端蛋白，通过残留的二硫键与较小的 C 端结构域连接。这些裂解的 TIMP2/4 复合物显示出扰乱的 MMP 抑制活性，说明 MMP9 可能对组织中的 TIMP:MMP 平衡具有特别显着的影响。