非血红素铁依赖性双加氧酶2-氨基乙硫醇双加氧酶(ADO)最近已被鉴定为酶性氧传感器,可通过调节带有N末端半胱氨酸(Nt-cys)的蛋白质的稳定性来协调细胞对缺氧的变化。N-degron途径。它催化Nt-Cys亚磺酰化,促进目标的O2依赖性蛋白酶体降解。只有少数ADO衬底得到了验证,包括G蛋白信号(RGS)4和5的调节因子,以及促炎细胞因子白介素32(IL32),所有这些都表现出细胞和/或组织特异性表达模式。ADO,相比之下,无处不在地表达,表明它可以以O2依赖性方式调节其他Nt-cys蛋白的稳定性。此外,单个化学基团的作用,活性位点金属,氨基酸组成和球状结构在蛋白质底物上的缔合仍然难以捉摸。为了帮助识别新的目标并检查系统的潜在生物化学,我们进行了一系列生物物理实验以研究已建立的ADO底物RGS5和IL32的结合要求。我们证明,使用表面等离子体激元反应(SPR)和酶测定,一个自由的,未修饰的Nt-硫醇和Nt-胺对于通过活性位点金属配位的底物接合至关重要,Nt-cys旁边的残基适度影响缔合和催化效率。此外,我们展示,通过1H-15N异核单量子相干(15N-HSQC)核磁共振(NMR)滴定,RGS5的球状部分对ADO关联的影响有限,与限制在N-末端的相互作用。这项工作建立了涉及ADO底物结合的关键特征,这将有助于识别新的蛋白质靶标,随后,阐明其在低氧适应中的作用。
The non-heme iron-dependent dioxygenase 2-aminoethanethiol dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyses Nt-cys sulfinylation, which promotes O2-dependent proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signalling (RGS) 4 and 5, and the pro-inflammatory cytokine interleukin-32 (IL32), all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. Furthermore, the role of individual chemical groups, active site metal, amino acid composition and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and IL32. We demonstrate, using surface plasmon response (SPR) and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence (15N-HSQC) nuclear magnetic resonance (NMR) titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.