Jobling Michael F, Mott Joni D, Finnegan Monica T, Jurukovski Vladimir, Erickson Anna C, Walian Peter J, Taylor Scott E, Ledbetter Steven, Lawrence Catherine M, Rifkin Daniel B, Barcellos-Hoff Mary Helen
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Radiat Res. 2006 Dec;166(6):839-48. doi: 10.1667/RR0695.1.
The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues.
三种哺乳动物转化生长因子β（TGF-β）亚型均以潜伏复合物的形式分泌，其中TGF-β同二聚体与各自称为潜伏相关肽（LAP）的前肽同二聚体非共价结合。TGF-β从其LAP中释放出来（称为激活）是TGF-β与细胞受体结合所必需的，因此细胞外激活是TGF-β生物利用度的关键调节点。我们之前的研究表明，潜伏性TGF-β1（LTGF-β1）在体内可被电离辐射有效激活，在体外可被芬顿化学产生的活性氧（ROS）激活。在本研究中，我们确定了使LTGF-β1对氧化还原敏感的特定ROS和蛋白质靶点。首先，我们比较了LTGF-β1、LTGF-β2和LTGF-β3，以确定这种激活机制的普遍性，发现氧化还原介导的激活仅限于LTGF-β1亚型。接下来，我们使用清除剂确定ROS激活是OH(.)可用性的函数，证实氧化是主要机制。为了确定LTGF-β1复合物的哪个伙伴在功能上被修饰，我们将每个伙伴暴露于ROS中，并测试其形成潜伏复合物的能力。TGF-β1的暴露并没有改变其与LAP结合的能力，但将LAP-β1暴露于ROS中则阻止了这种现象，而用温和的还原剂处理暴露于ROS的LAP-β1可恢复其中和TGF-β1活性的能力。综上所述，这些结果表明，LAP-β1中ROS诱导的氧化触发了构象变化，从而释放了TGF-β1。通过位点特异性突变，我们确定了LAP-β1特有的位于氨基酸位置253的甲硫氨酸残基对ROS介导的激活至关重要。我们提出，LTGF-β1包含一个以甲硫氨酸253为中心的氧化还原开关，这使得LTGF-β1能够独特地作为组织中氧化应激的细胞外传感器发挥作用。
