Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, USA.
Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut, USA.
J Cell Physiol. 2021 Nov;236(11):7745-7758. doi: 10.1002/jcp.30457. Epub 2021 Jun 1.
The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (K = 99 ± 19 μM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2 [Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.
许多参与体内平衡控制的肽的生物合成都需要肽基甘氨酸 α-酰胺化单加氧酶（PAM），这是一种古老的、高度保守的铜离子和抗坏血酸依赖性酶。使用氨肽酶 chromogranin A 的产生来监测肿瘤细胞中 PAM 的功能，发现生理相关水平的缺氧会抑制这种单加氧酶。暴露于缺氧条件下 4 小时的原代垂体细胞产生氨肽酶 chromogranin A 的能力也受到类似抑制。纯化的单加氧酶对氧气的亲和力（K = 99 ± 19 μM）与这一结果一致。PAM 在正常氧条件下改变分泌途径行为的能力需要其单加氧酶活性。在正常氧条件下，表达高水平 PAM 的促肾上腺皮质激素肿瘤细胞密集培养物中的缺氧诱导因子 1a 水平超过对照细胞；表达无活性的单加氧酶没有这种效果。缺氧对两种 PAM 调节基因（激活转录因子 3 [Atf3]和 FK506 结合蛋白 2 [Fkbp2]）的水平的影响在表达高水平和低水平 PAM 的细胞中不同。人类和小鼠 PAM 中都存在假定的缺氧反应元件，hPAM 一直被鉴定为对缺氧反应上调的基因之一。PAM 的表达也已知会改变基因表达。在对缺氧反应上调的基因中，有四分之一的基因在 PAM 表达增加后下调。综上所述，我们的数据表明 PAM 和氨肽酶分泌在组织特异性缺氧反应的协调中起作用。
