The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.     

SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.     

生命的低氧适应——2019年度诺贝尔生理学或医学奖成果解析

 氧的利用和调节是高等生命赖以生存的基本条件，威廉·凯林、彼得·拉特克利夫和格雷格·塞门扎3位科学家因发现细胞感知和适应氧气供应的相关机制而获得了2019年度诺贝尔生理学或医学奖。他们发现低氧诱导因子1（hypoxia-inducible factors 1，HIF-1）广泛存在于急、慢性缺氧细胞中，是细胞适应低氧的重要转录因子。HIF-1水平受氧气含量的调节。高氧条件下，HIF-1被修饰进而降解；低氧条件下，HIF-1不被降解，并通过转录调节引起促红细胞生成素等低氧相关基因的表达。本文通过介绍HIF-1的发现和基本分子机制，探讨其在临床中的应用价值。     

An oxygen-regulated switch in the protein synthesis machinery

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m(7)-GpppG) 5'?cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α-RBM4-eIF4E2 complex captures the 5'?cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.     

HIF-1-modified BMSCs improve migration and reduce neuronal apoptosis after stroke in rats

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated ameliorating neurologic deficits after stroke. Hypoxia-inducible factor-1 (HIF-1), the key regulator of cellular responses to low oxygen concentration, can activate multiple genes involving in crucial aspects for neurologic recovery. In this study, we present that rat BMSCs overexpression of HIF-1 α showed higher expression of HIF-1 target genes in HIF-1-BMSCs, including CXCR4, EPO, and VEGF. BMSCs-mHIF-1α also exhibited an enhanced mobility towards the ischemic area within rat brain. Neural cell apoptosis in ischemic brain shown less severe in rats transplanted with HIF-1-BMSCs. Furthermore, the number of cells expressing neural progenitor markers PAX6 and DCX were increased in BMSCs-mHIF-1α-transplanted rats. These results suggest that HIF-1α in BMSCs reduces neuronal apoptosis and promotes neurogenesis after stroke in rats.     

细胞低氧感知与响应——2019年诺贝尔生理学或医学奖成果简析

 2019年诺贝尔生理学或医学奖颁给了William G.Kaelin、Jr、Sir Peter J.Ratcliffe、Gregg L.Semenza，以奖励他们在细胞低氧感知与适应的相关研究中所做出的开创性贡献。他们发现细胞利用了转录因子HIF-1的α亚基上的羟基化修饰来感知细胞内的氧水平，而低氧条件下未羟基化的HIF-1α会快速累积，并进入细胞核内调控上千个基因的转录。细胞低氧响应与多种人类疾病紧密相关，包括贫血症、红细胞增多症、心血管疾病、卒中以及癌症。回顾了细胞低氧响应信号通路的发现过程以及与低氧响应有关的人类疾病。     

SUMO-1在ApoE-/-小鼠PM2.5暴露中调控血管HIF-1α/VEGF信号通路的研究

本文研究了ApoE-/-小鼠经PM2.5暴露后,其主动脉弓中SUMO-1、HIF-1及其靶基因血管内皮生长因子（VEGF）表达变化规律及HIF-1的SUMO化修饰情况。利用气管滴注方法将颗粒物滴入ApoE-/-小鼠,利用Western blot、QPCR检测小鼠主动脉中SUMO-1、HIF-1、VEGF的表达情况和CO-IP法检测SUMO-1及HIF-1共修饰情况。 结果显示: PM2.5暴露后,小鼠主动脉SUMO-1、HIF-1、VEGF的表达上调。CO-IP 显示:对照组SUMO-1没有对HIF-1α发生SUMO化修饰,PM2.5暴露后SUMO-1对HIF-1α产生了明显的SUMO化修饰。SUMO化的HIF-1与HIF-1α、HIF-1、VEGF蛋白均呈正相关。 PM2.5暴露可能通过上调细胞中SUMO-1的表达,稳定HIF-1α或者上调HIF-1的表达,进而影响VEGF的表达。     

转录调节因子DEC1的研究进展

DEC1（Differentiated embryo-chondrocyte expressed gene1）是一个碱性螺旋一环一螺旋（bHLH）结构的转录因子,在软骨形成、神经发生、免疫应答、分子钟的调控、细胞分化、肿瘤的发生中起着重要的作用。DEC1还是一个缺氧调节基因,与肿瘤细胞在缺氧环境下存活密切相关,可调控肿瘤细胞生长、凋亡相关因子如缺氧诱导因子1α、转化生长因子-β、信号转导和转录激活因子、P53等的表达。DEC1还可通过调节同一家族的分子如DEC2的表达来调节细胞的功能。本文就其研究进展加以概述。     

Genetically modified photosynthetic antenna complexes with blueshifted absorbance bands.

Light energy for photosynthesis is collected by the antenna system, creating an excited state which migrates energetically 'downhill'. To achieve efficient migration of energy the antenna is populated with a series of pigments absorbing at progressively redshifted wavelengths. This variety in absorbing species in vivo has been created in a biosynthetically economical fashion by modulating the absorbance behaviour of one kind of (bacterio)chlorophyll molecule. This modulation is poorly understood but has been ascribed to pigment-pigment and pigment-protein interactions. We have examined the relationship between aromatic residues in antenna polypeptides and pigment absorption, by studying the effects of site-directed mutagenesis on a bacterial antenna complex. A clear correlation was observed between the absorbance of bacteriochlorophyll a and the presence of two tyrosine residues, alpha Tyr44 and alpha Tyr45, in the alpha subunit of the peripheral light-harvesting complex of Rhodobacter sphaeroides, a purple photosynthetic bacterium that provides a well characterized system for site-specific mutagenesis. By constructing single (alpha Tyr44, alpha Tyr45----PheTyr) and then double (alpha Tyr44, alpha Tyr45----PheLeu) site-specific mutants, the absorbance of bacteriochlorophyll was blueshifted by 11 and 24 nm at 77 K, respectively. The results suggest that there is a close approach of tyrosine residues to bacteriochlorophyll, and that this proximity may promote redshifts in vivo.     

PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1alpha (HIF-1alpha) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis.     

Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.     

Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.     

青藏高原短居人群缺氧风险性评价

青藏高原具有鲜明的高寒缺氧的气象特征,对短居人群的健康有严重影响,从自然地理的角度研究青藏高原缺氧风险对地方发展与缺氧政策制定具有重要意义。基于2021年7月采集青藏高原不同海拔地区的气压、含氧量和短居人群的血氧饱和度等数据,建立了海拔与血氧饱和度的关系,绘制了青藏高原短居缺氧空间分布图。结果表明：(1)随着海拔的升高,绝对含氧量线性下降(y=-0.0325x+280.45,n=70,r²=0.94),绝对含氧量与海拔呈线性关系。(2)随着海拔的升高,血氧饱和度呈指数下降,缺氧风险呈指数上升(y=104-0.68×e^0.35x+1.77,n=70,r²=0.57)。(3)根据血氧饱和度与海拔高度的关系,青藏高原缺氧低风险区、缺氧中风险区和缺氧高风险区占青藏高原总面积比分别为10.6%、32.0%和57.4%,其中低风险区主要分布在青海东北部、柴达木盆地和林芝市以南,中风险区分布在青海西北部、西藏东部山地和青藏高原河流谷地,高风险区主要分布在藏北高原无人区和喜马拉雅山系附近。