低氧诱导因子HIF-1是低氧应答的关键调节因子，其脯氨酸羟化酶的识别基序LXXLAP高度保守，我们发现青海湖裸鲤是目前已知的唯一一种在该基序发生变异的脊椎动物，并可能导致其HIF-1α在常氧下相对稳定。青海湖湖水较高的pH会使鱼类的氨氮排泄困难并出现氨中毒，青海湖裸鲤对碱性水环境的适应机制尚不清楚。推测HIF-1α的相对稳定，对于裸鲤适应氨中毒造成的"代谢性低氧"，减少氨氮的产生和避免线粒体损伤具有重要作用。本项目研究这一变异对裸鲤HIF-1α羟化水平和稳定性的影响，以及对裸鲤在不同水环境中代谢途径的调节作用，和高氨环境下对裸鲤细胞线粒体结构的保护机制，以明确该变异对于青海湖裸鲤适应高原盐湖碱性环境的生理意义，提出一种鱼类耐氨的新分子机制。

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. Under normoxia, HIF-1α is hydroxylated in a PHD-dependent manner, which leads to its ubiquitination by the von Hippel-Lindau protein (VHL) and proteasomal degradation. Under hypoxia, the hydroxylase activity of PHD enzymes is inhibited, thereby allowing the stable formation of the heterodimeric HIF transcription factor and its activation. Both proline residues subjected to hydroxylation lie within the LXXLAP amino acid motif in the oxygen-dependent degradation domains, conserved between the worm and the mammalian HIF-1α isoforms except for the second praline of naked carp HIF-1α, which has the sequence PEMLAP.The naked carp (Gymnocypris przewalskii) resides in the alkaline (pH ~9.3) and saline waters of Lake Qinghai. The response of fish to high environmental pH has been the occurrence of inhibited ammonia-N excretion, accompanied by elevated internal levels of ammonia-N. The mechanisms to protect naked carp from extremely alkaline environment are still unknown. Mobility shift assay, pVHL capture assay, MALDI-TOF MS analysis and Western blot will be used to study the influence of this sequence change to the hydroxylation levels and stability of naked carp HIF-1α. By gene chip technology combined with the use of qPCR and enzyme activity assay we may be able to determine how HIF-1α acts to influence the metabolic pathway of naked carp. Calcein fluorescence studies will be carried out to demonstrate the protection of HIF-1α to mitochondrial structure and function in hyperammonia stress. The aim of this study is to identify whether the sequence change in HIF-1α of naked carp contribute to physiological adaptation to the alkaline water of Lake Qinghai. It is likely that this study would verify a new adaptation used by naked carp to defend against ammonia toxicity.

项目主要研究青海湖裸鲤HIF-1α P550变异对HIF-1α稳定性和羟化水平的影响，是否参与动物对高原盐湖碱性环境适应的调节，以及在高氨诱导细胞线粒体损伤过程中的可能保护作用与机制。研究表明，与在其它脊椎动物中保守的L550相比，P550变异使青海湖裸鲤HIF-1α羟化水平降低，不容易被pVHL识别并结合进入泛肽降解途径，具有较高的稳定性。研究表明HIF-1α参与青海湖裸鲤对高原盐湖碱性环境的应答，并可有效降低碱性环境中血氨的积累。转录组和qPCR等分析发现，不同HIF-1水平影响裸鲤糖酵解-三羧酸循环途径PFK (Phosphofructokinase)和PDK1 (Pyruvate dehydrogenase kinase 1)，谷氨酰胺代谢途径GS (Glutamine synthetase)和GDH (Glutamate dehydrogenase)的表达，以及GPT (Glutamic pyruvic transaminase)酶活和NADPH的合成。NH4Cl处理抑制CIK细胞增殖，并诱导活性氧水平上升和细胞凋亡，而DMOG协同处理可消除这一影响。表明HIF-1α可通过调节线粒体的三羧酸循环和谷氨酰胺代谢，减少氨氮的产生，降低高氨诱导的活性氧的生成，从而降低对线粒体的损伤，改善碱性环境中鱼类对体内高氨水平的适应。