Abstract 抽象
The increase in body size of humans and other vertebrates requires a physiological infrastructure to provide adequate delivery of oxygen to tissues and cells to maintain oxygen homeostasis. The heart, lungs and the vasculature are all part of a highly regulated system that ensures the distribution of the precise amount of oxygen needed throughout the mammalian organism. Given its fundamental impact on physiology and pathology, it is no surprise that the response of cells to a lack of oxygen, termed hypoxia, has been the focus of many research groups worldwide for many decades now. The transcriptional complex hypoxia-inducible factor has emerged as a key regulator of the molecular hypoxic response, mediating a wide range of physiological and cellular mechanisms necessary to adapt to reduced oxygen.
人类和其他脊椎动物体型的增加需要一种生理基础设施,为组织和细胞提供足够的氧气输送,以维持氧稳态。心脏、肺和脉管系统都是高度调节系统的一部分,该系统确保在整个哺乳动物生物体中分配所需的精确氧气量。鉴于其对生理学和病理学的根本影响,细胞对缺氧的反应(称为缺氧)几十年来一直是全球许多研究小组的关注点也就不足为奇了。转录复合物缺氧诱导因子已成为分子缺氧反应的关键调节因子,介导适应减少氧气所需的广泛生理和细胞机制。
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Molecular Biology of Hypoxia-Inducible Factor 1 (HIF-1)
缺氧诱导因子 1 (HIF-1) 的分子生物学
Investigation of the molecular mechanisms of one of the most striking responses to hypoxia – the induction of the hematopoietic growth hormone erythropoietin (EPO) – paved the way for the first identification of a hypoxia-inducible transcription factor. When the blood oxygen content is reduced in anemia or at high altitude, the EPO production in renal interstitial fibroblasts is rapidly turned on. The up to several 100-fold induction of EPO mRNA and protein induces erythropoietic responses that directly increase blood oxygen transport. Studies of DNA–protein interactions at the 3′ enhancer of the EPO gene identified a protein complex that only bound in hypoxia. It was designated hypoxia-inducible factor 1 (HIF-1) by Semenza and Wang.1
对缺氧最引人注目的反应之一的分子机制的研究 - 造血生长激素促红细胞生成素 (EPO) 的诱导 - 为首次鉴定缺氧诱导转录因子铺平了道路。当贫血或高海拔地区血氧含量降低时,肾间质成纤维细胞中的 EPO 产生迅速启动。EPO mRNA 和蛋白质的高达 100 倍的诱导诱导红细胞生成反应,直接增加血氧运输。对 EPO 基因 3' 增强子处的 DNA-蛋白质相互作用的研究确定了一种仅在缺氧中结合的蛋白质复合物。它被 Semenza 和 Wang 命名为缺氧诱导因子 1 (HIF-1)。1
It soon became obvious that the HIF system is a key regulator of a broad range of cellular and systemic responses to hypoxia and acts in all mammalian cells. Changes in gene expression directly or indirectly regulated by HIF extend to well over a 100 genes. HIF-mediated pathways influence metabolic adaptation, erythropoiesis, angiogenesis and vascular tone, cell growth and differentiation, survival and apoptosis, and thus are critical factors in development, physiology and disease (for review, see Maxwell et al.2).
很快就发现,HIF 系统是对缺氧的广泛细胞和全身反应的关键调节因子,并作用于所有哺乳动物细胞。受 HIF 直接或间接调节的基因表达变化延伸到 100 多个基因。HIF 介导的通路影响代谢适应、红细胞生成、血管生成和血管张力、细胞生长和分化、存活和细胞凋亡,因此是发育、生理学和疾病的关键因素(综述见 Maxwell 等人。2).
HIF is a heterodimeric DNA-binding complex composed of two basic helix-loop-helix proteins of the PAS family (PER, AHR, ARNT and SIM family):3 the constitutive HIF-1β and one of either hypoxia-inducible α-subunits, HIF-1α or HIF-2α. In hypoxia, the α/β heterodimer binds to a core pentanucleotide sequence (RCGTG) in the hypoxia response elements (HREs) of target genes. HIF-β subunits are non-oxygen-responsive nuclear proteins that also have other roles in transcription, for example, in the xenobiotic response (for review, see Gu et al.4). In contrast, the HIF-α subunits are highly inducible by hypoxia.
HIF 是一种异二聚体 DNA 结合复合物,由 PAS 家族(PER、AHR、ARNT 和 SIM 家族)的两个碱性螺旋-环-螺旋蛋白组成:3组成型 HIF-1β和缺氧诱导型 α 亚基之一,HIF-1α 或 HIF-2α。在缺氧时,α/β 异二聚体与靶基因缺氧反应元件 (HRE) 中的核心五核苷酸序列 (RCGTG) 结合。HIF-β 亚基是非氧反应性核蛋白,在转录中也具有其他作用,例如,在外源性反应中(综述,参见 Gu 等人。相比之下,HIF-α 亚基高度易受缺氧诱导。
Regulation of HIF-α Subunits by Protein Hydroxylation
蛋白质羟基化对 HIF-α 亚基的调控
Under normoxic conditions, HIF-α subunits have a very short half-life.5 Cells continuously synthesize and degrade HIF-α protein. However, under decreasing concentrations of oxygen, the degradation of HIF-α is retarded.6 The interface between oxygen and the HIF-α subunit is provided by distinct enzymatic reactions: the hydroxylation of two prolyl residues (Pro402 and Pro564 in human HIF-1α) in the oxygen-dependent degradation domain (ODDD) of the α-subunits.7, 8 This oxygen-dependent hydroxylation regulates the interaction with the von Hippel–Lindau tumor suppressor protein (pVHL). pVHL is the recognition component of an E3 ubiquitin ligase complex that targets HIF-α for proteolysis by the ubiquitin–proteasome pathway.9, 10 Other components of the complex are elongin B, elongin C, Rbx1 and Cul2, which also participate in other E3 ubiquitin ligase complexes. Under hypoxic conditions, prolyl hydroxylation is suppressed, HIF-α protein escapes proteasomal destruction and can accumulate. It translocates to the nucleus and dimerizes with HIF-1β. The heterodimeric transactivating complex HIF then binds to the HRE in promoter or enhancer sequences of target genes (Figure 1).
在常氧条件下,HIF-α 亚基的半衰期非常短。5 细胞持续合成和降解 HIF-α 蛋白。然而,在氧气浓度降低的情况下,HIF-α 的降解被延缓。6 氧和 HIF-α 亚基之间的界面由不同的酶促反应提供:α 亚基的氧依赖性降解结构域 (ODDD) 中两个脯氨酰残基(人 HIF-1α中的 Pro402 和 Pro564)的羟基化。7、8这种氧依赖性羟基化调节与 von Hippel-Lindau 抑癌基因蛋白 (pVHL) 的相互作用。pVHL 是 E3 泛素连接酶复合物的识别组分,该复合物靶向 HIF-α,通过泛素-蛋白酶体途径进行蛋白水解。9、10该复合物的其他组分是 elongin B、elongin C、Rbx1 和 Cul2,它们也参与其他 E3 泛素连接酶复合物。在缺氧条件下,脯氨酰羟基化受到抑制,HIF-α 蛋白逃避蛋白酶体破坏并可以积累。它易位到细胞核并与 HIF-1β二聚化。然后,异二聚体反式激活复合物 HIF 与靶基因启动子或增强子序列中的 HRE 结合(图 1)。
The HIF-modifying enzymes were identified as related to egl-9 in Caenorhabditis elegans and termed prolyl hydroxylase domain (PHD) enzymes (PHD1–PHD3).11, 12 The PHDs are non-heme Fe(II)- and 2-oxoglutarate-dependent dioxygenases that split molecular oxygen. One oxygen atom is inserted into the HIF-α peptide at the prolyl residue, the other reacts with 2-oxoglutarate yielding succinate and CO2 as products. In hypoxia, the HIF prolines remain unmodified. The effects of hypoxia can be mimicked by iron chelation, use of 2-oxoglutarate analogs such as dimethyloxalylglycine or substitution of Fe(II) by metal ions such as cobalt.
HIF 修饰酶被鉴定为与秀丽隐杆线虫中的 egl-9 相关,称为 prolyl hydroxylase domain (PHD) 酶 (PHD1-PHD3)。11、12PHD 是非血红素 Fe(II) 和 2-氧代戊二酸依赖性双加氧酶,可分裂分子氧。一个氧原子在脯氨酰残基处插入 HIF-α 肽中,另一个氧原子与 2-氧代戊二酸反应,产生琥珀酸盐和 CO2 作为产物。在缺氧时,HIF 脯氨酸保持不变。缺氧的影响可以通过铁螯合、使用 2-氧代戊二酸类似物(如二甲基草酰甘氨酸)或用金属离子(如钴)取代 Fe(II) 来模拟。
The ODDD of the HIF-α subunit contains an N-terminal (N-ODDD) and a C-terminal portion (C-ODDD),13, 14 both of which can interact with pVHL independently.15 Furthermore, two transactivation domains (TADs) have been defined in HIF-1α and HIF-2α: an N-terminal TAD (which overlaps with the C-ODDD) and a C-terminal TAD.16 In contrast to regulation of HIF-α stability by proline modification in the ODDD, transcriptional activity is regulated by the hydroxylation of a C-terminal asparagine residue (Asn 803 in human HIF-1α).17 The hydroxylation reaction is carried out by an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) and this modification prevents interaction of the HIF-α C-TAD with the CH-1 domain of the transcriptional coactivator p300.18, 19 Thus, in contrast to the prolyl hydroxylation that enables protein–protein interaction, the asparaginyl hydroxylation prevents protein recruitment. FIH can also interact with pVHL,20 which fits well with the observation that an almost complete upregulation of HIF target genes can be found in cells devoid of functional pVHL.9, 21 This suggests that in the absence of pVHL both the oxygen-dependent controls of stability and activity of HIF-α are diminished and thus the functions of VHL might not be limited to those of an E3 ligase.
HIF-α 亚基的 ODDD 包含一个 N 端 (N-ODDD) 和一个 C 端部分 (C-ODDD),13, 14,两者都可以独立地与 pVHL 相互作用。15 此外,HIF-1α 和 HIF-2α中定义了两个反式激活结构域 (TAD):N 端 TAD(与 C-ODDD 重叠)和 C 端 TAD。16 与 ODDD 中脯氨酸修饰对 HIF-α 稳定性的调节相反,转录活性受 C 末端天冬酰胺残基(人 HIF-1α中的 Asn 803)的羟基化调节。17 羟基化反应由称为 f actor-i的天冬酰胺羟化酶抑制 HIF (FIH) 进行,这种修饰阻止了 HIF-α C-TAD 与转录共激活因子 p300 的 CH-1 结构域的相互作用。18、19因此,与实现蛋白质-蛋白质相互作用的脯氨酰羟基化相反,天冬酰胺羟基化阻止了蛋白质募集。FIH 还可以与 pVHL 相互作用,