BACKGROUND & AIMS: :The hypoxia-inducible factor 1alpha (HIF-1alpha) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1alpha is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1alpha in normoxia. Here, we describe a new regulator of HIF-1alpha, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1alpha leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1alpha in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1alpha levels in normoxia and hypoxia in both pVHL-competent and -deficient cells, whereas HAF knockdown increases HIF-1alpha levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2alpha is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1alpha accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1alpha-specific degradation pathway that degrades HIF-1alpha through a new, oxygen-independent mechanism.

背景与目标： ：缺氧诱导因子1alpha（HIF-1alpha）是细胞对缺氧反应的主要调节因子。 HIF-1alpha的关键调控因子是von Hippel-Lindau蛋白（pVHL），它介导常氧条件下HIF-1alpha的氧依赖性蛋白酶体降解。在这里，我们描述了一种新的HIF-1alpha调节因子，缺氧相关因子（HAF），一种新型的E3-泛素连接酶，它结合HIF-1alpha导致其蛋白酶体依赖性降解，而与细胞氧张力无关。 HAF是一种在增殖细胞中表达的蛋白质，在体外结合并泛素化HIF-1alpha，结合和E3连接酶活性均由HAF氨基酸654-800介导。此外，HAF的过表达降低了正常氧和低氧状态下HIF-1alpha的水平。具有pVHL能力的细胞和缺陷型细胞，而HAF敲低会增加常氧，低氧和表皮生长因子刺激下的HIF-1alpha水平。相反，HIF-2alpha不受HAF调控。在体内，来自过表达HAF的细胞的异种移植物显示HIF-1alpha的水平降低，并伴随着肿瘤的生长和血管生成的降低。因此，HAF是新的HIF-1alpha特异性降解途径的关键介体，该途径通过新的，与氧无关的机制降解HIF-1alpha。

BACKGROUND & AIMS: :Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A in vivo. The majority of DDB2 and CUL4A diffuse in the nucleus with a diffusion rate consistent with a high molecular mass complex. Essentially all DDB2 binds to UV-induced DNA damage, where each molecule resides for approximately 2 minutes. After the induction of DNA damage, DDB2 is proteolytically degraded with a half-life that is two orders of magnitude larger than its residence time on a DNA lesion. This indicates that binding to damaged DNA is not the primary trigger for DDB2 breakdown. The bulk of DDB2 binds to and dissociates from DNA lesions independently of damage-recognition protein XPC. Moreover, the DDB2-containing E3 ubiquitin ligase is bound to many more damaged sites than XPC, suggesting that there is little physical interaction between the two proteins. We propose a scenario in which DDB2 prepares UV-damaged chromatin for assembly of the NER complex.

背景与目标： DNA损伤蛋白结合蛋白2（DDB2）对紫外线损伤的DNA具有高亲和力，并与哺乳动物的全基因组核苷酸切除修复（NER）的初始步骤有关。 DDB2与CUL4A结合并形成E3泛素连接酶。在这项研究中，我们已经分析了DDB2和CUL4A在体内的特性。 DDB2和CUL4A的大部分以与高分子复合物一致的扩散速率扩散到细胞核中。基本上所有DDB2都与紫外线诱导的DNA损伤结合，每个分子在其中停留约2分钟。诱导DNA损伤后，DDB2被蛋白水解降解，其半衰期比其在DNA病变上的停留时间长两个数量级。这表明与损坏的DNA结合不是DDB2分解的主要诱因。 DDB2的大部分独立于损伤识别蛋白XPC与DNA损伤结合和解离。此外，与XPC相比，含DDB2的E3泛素连接酶结合的损伤位点更多，这表明这两种蛋白之间几乎没有物理相互作用。我们提出了一种方案，其中DDB2制备UV损伤的染色质以组装NER复合物。

BACKGROUND & AIMS: :Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP)--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP) E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

背景与目标： ：已显示人类免疫缺陷病毒1型（HIV-1）病毒蛋白R（Vpr）通过诱导ATR介导的p34cdc2失活而导致人细胞中的G2细胞周期停滞，但直接参与这一过程的因素仍然未知。我们使用串联亲和纯化分离天然Vpr复合物。我们发现受损的DNA结合蛋白1（DDB1），病毒蛋白R结合蛋白（VPRBP）和cullin 4A（CUL4A）是CUL4A E3泛素连接酶复合物DDB1-CUL4A（VPRBP）的组成部分与Vpr。小干扰RNA消耗VPRBP会损害Vpr介导的G2阻滞诱导。重要的是，仅VPRBP敲低并不会影响正常的细胞周期进程或ATR检查点的激活，这表明VPRBP参与G2阻滞是Vpr特有的。此外，亮氨酸/富含异亮氨酸的域Vpr突变体与VPRBP和DDB1相互作用的能力受损，也导致G2阻滞作用大大减弱。相比之下，发现G2逮捕缺陷的C末端Vpr突变体保持它们与这些蛋白质缔合的能力，这表明Vpr与DDB1-VPRBP复合物的相互作用是必要的，但不足以阻止细胞周期进程。总体而言，这些结果指向一个模型，其中Vpr可以充当DDB1-CUL4A（VPRBP）E3泛素连接酶复合物与未知细胞因子之间的连接子，该细胞因子通过泛素化进行蛋白水解或活性调节将激活ATR介导的检查点信号并诱导G2逮捕。

BACKGROUND & AIMS: :Drought stress conditions in soil or air hinder plant growth and development. Here, we report that the hot pepper (C apsicum a nnuum) RING type E3 Ligase 1 gene (CaREL1) is essential to the drought stress response. CaREL1 encodes a cytoplasmic- and nuclear-localized protein with E3 ligase activity. CaREL1 expression was induced by abscisic acid (ABA) and drought. CaREL1 contains a C3H2C3-type RING finger motif, which functions in ubiquitination of the target protein. We used CaREL1-silenced pepper plants and CaREL1-overexpressing (OX) transgenic Arabidopsis plants to evaluate the in vivo function of CaREL1 in response to drought stress and ABA treatment. CaREL1-silenced pepper plants displayed a drought-tolerant phenotype characterized by ABA hypersensitivity. In contrast, CaREL1-OX plants exhibited ABA hyposensitivity during the germination, seedling, and adult stages. In addition, plant growth was severely impaired under drought stress conditions, via a high level of transpirational water loss and decreased stomatal closure. Quantitative RT-PCR analyses revealed that ABA-related drought stress responsive genes were more weakly expressed in CaREL1-OX plants than in wild-type plants, indicating that CaREL1 functions in the drought stress response via the ABA-signalling pathway. Taken together, our results indicate that CaREL1 functions as a negative regulator of ABA-mediated drought stress tolerance.

背景与目标： ：土壤或空气中的干旱胁迫条件会阻碍植物的生长和发育。在这里，我们报告辣椒（C apsicum anuum）RING型E3连接酶1基因（CaREL1）对干旱胁迫响应至关重要。 CaREL1编码具有E3连接酶活性的胞质和核定位蛋白。 CaREL1表达是由脱落酸（ABA）和干旱诱导的。 CaREL1包含一个C3H2C3型RING手指基序，在目标蛋白的泛素化中起作用。我们使用CaREL1沉默的辣椒植物和CaREL1过表达（OX）转基因拟南芥植物来评估CaREL1在体内响应干旱胁迫和ABA处理的功能。 CaREL1沉默的辣椒植物表现出以ABA超敏性为特征的耐旱表型。相反，CaREL1-OX植物在发芽，幼苗和成年阶段均表现出ABA低敏感性。另外，由于高水平的蒸腾失水和减少的气孔关闭，在干旱胁迫条件下植物的生长受到严重损害。定量RT-PCR分析显示，与野生型植物相比，CaREL1-OX植物中与ABA相关的干旱胁迫响应基因的表达更弱，表明CaREL1通过ABA信号通路在干旱胁迫响应中发挥功能。两者合计，我们的结果表明CaREL1充当ABA介导的干旱胁迫耐受性的负调节剂。

BACKGROUND & AIMS: :Polycomb proteins are critical chromatin modifiers that regulate stem cell differentiation via transcriptional repression. In skeletal muscle progenitors Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), contributes to maintain the chromatin of muscle genes in a repressive conformation, whereas its down-regulation allows the progression through the myogenic programme. Here, we show that p38α kinase promotes EZH2 degradation in differentiating muscle cells through phosphorylation of threonine 372. Biochemical and genetic evidence demonstrates that the MYOD-induced E3 ubiquitin ligase Praja1 (PJA1) is involved in regulating EZH2 levels upon p38α activation. EZH2 premature degradation in proliferating myoblasts is prevented by low levels of PJA1, its cytoplasmic localization and the lower activity towards unphosphorylated EZH2. Our results indicate that signal-dependent degradation of EZH2 is a prerequisite for satellite cells differentiation and identify PJA1 as a new player in the epigenetic control of muscle gene expression.

背景与目标： ：Polycomb蛋白是重要的染色质修饰剂，可通过转录抑制调节干细胞分化。在骨骼肌祖细胞中，zeste同源物2（EZH2）的增强子是Polycomb Repressive Complex 2（PRC2）的催化亚基，有助于将肌肉基因的染色质维持在抑制状态，而其下调则允许通过成肌程序进行。在这里，我们显示p38α激酶通过苏氨酸372的磷酸化促进分化肌肉细胞中的EZH2降解。生化和遗传证据表明，MYOD诱导的E3泛素连接酶Praja1（PJA1）在p38α激活后参与调节EZH2的水平。 EZH2在增殖的成肌细胞中过早降解被低水平的PJA1，其细胞质定位和对未磷酸化EZH2的较低活性所阻止。我们的结果表明，EZH2的信号依赖性降解是卫星细胞分化的先决条件，并确定PJA1是肌肉基因表达的表观遗传控制中的新角色。

BACKGROUND & AIMS: :Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

背景与目标： 泛素E3连接酶的缺陷与多种人类疾病（包括癌症）的发病机制有关，因为它们在控制多种信号通路中起着核心作用。 RING E3连接酶可促进多种细胞事件必不可少的蛋白质的泛素化。鉴定哪些泛素连接酶特异性地影响不同的细胞过程，对于开发针对这些疾病的靶向疗法至关重要。在这里，我们讨论两种新型的RING E3连接酶BCA2和RNF11，它们与人乳腺癌密切相关。 BCA2 E3连接酶与雌激素受体共调节，并在表皮生长因子受体（EGF-R）转运的调节中发挥作用。 RNF11是一个小的RING E3连接酶，可影响转化生长因子β和EGF-R信号传导，并在浸润性乳腺癌中过表达。这两种蛋白证明了RING E3连接酶相互作用在乳腺癌中的复杂性，是治疗干预的潜在靶标。

BACKGROUND & AIMS: :The bean pathogen Pseudomonas syringae pv. syringae B728a expresses homologs of the type III effectors AvrPto and AvrPtoB, either of which can trigger resistance in tomato cultivars expressing Pto and Prf genes. We found that strain B728a also elicits nonhost resistance in tomato cultivars VFNT Cherry and Moneymaker that lack Pto but express other members of the Pto family (e.g., SlFen and SlPtoC). Here, we show that the AvrPtoB homolog from B728a, termed AvrPtoBB728a (also known as HopAB1), is recognized by 'VFNT Cherry' and 'Moneymaker' when the effector is expressed in P. syringae pv. syringae 61, a strain lacking the avrPto or avrPtoB homolog. Using a gene-silencing approach, this recognition was shown to involve one or more Pto family members and Prf. AvrPtoBB728a interacted with SlFen, SlPtoC, and SlPtoD, in addition to Pto, in a yeast two-hybrid assay. In P. syringae pv. tomato DC3000, the C-terminal domain of AvrPtoB is an E3 ubiquitin ligase that ubiquitinates Fen, causing its degradation and leading to disease susceptibility. Although the C-terminal domain of AvrPtoBB728a shares 69% amino acid identity with that of AvrPtoB, we found that it has greatly reduced E3 ligase activity and is unable to ubiquitinate Fen in an in vitro ubiquitination assay. Thus, the nonhost resistance of 'VFNT Cherry' and 'Moneymaker' to B728a appears to be due to recognition of AvrPtoBB728 as a result of the effector's reduced E3 ligase activity, which prevents it from facilitating degradation of a Pto family member. We speculate that the primary plant host of B728a lacks a Fen-like protein and that, therefore, the E3 ligase of AvrPtoBB728 was unnecessary for pathogenicity and has diverged and become ineffective.

背景与目标： ：豆病原体丁香假单胞菌PV。丁香香脂素B728a表达III型效应子AvrPto和AvrPtoB的同源物，它们两者均可触发表达Pto和Prf基因的番茄品种的抗性。我们发现菌株B728a在缺乏Pto但表达Pto家族其他成员的番茄品种VFNT Cherry和Moneymaker中也引起了非寄主抗性。在这里，我们显示了当在丁香假单胞菌pv中表达效应子时，来自B728a的AvrPtoB同源物，称为AvrPtoBB728a（也称为HopAB1），被“ VFNT Cherry”和“ Moneymaker”识别。丁香61，缺乏avrPto或avrPtoB同源物的菌株。使用基因沉默方法，该识别显示涉及一个或多个Pto家族成员和Prf。在酵母双杂交试验中，AvrPtoBB728a除Pto外还与SlFen，SlPtoC和SlPtoD相互作用。在丁香假单胞菌pv。番茄DC3000，AvrPtoB的C末端域是E3泛素连接酶，泛素化Fen，导致其降解并导致疾病易感性。尽管AvrPtoBB728a的C末端结构域与AvrPtoB具有69％的氨基酸同一性，但我们发现它具有大大降低的E3连接酶活性，并且在体外泛素化测定中无法泛素化Fen。因此，“ VFNT Cherry”和“ Moneymaker”对B728a的非宿主抗药性似乎是由于效应子E3连接酶活性降低导致对AvrPtoBB728的识别，从而阻止了它促进Pto家族成员的降解。我们推测B728a的主要植物宿主缺乏Fen样蛋白，因此，AvrPtoBB728的E3连接酶对于致病性而言是不必要的，并且已经分化并且变得无效。

BACKGROUND & AIMS: :The forkhead transcription factor, DAF-16, a downstream target of the insulin/IGF-I signaling pathway in C. elegans, is indispensable both for lifespan regulation and stress resistance. The molecular mechanisms involved in regulating DAF-16 transcriptional activation remain undefined. Here, we have identified an E3 ubiquitin ligase, RLE-1 (regulation of longevity by E3), which regulates aging in C. elegans. Disruption of RLE-1 expression in C. elegans increases lifespan; this extension of lifespan is due to elevated DAF-16 protein but not to changes of daf-16 mRNA levels. We have also found that RLE-1 catalyzes DAF-16 ubiquitination, leading to degradation by the proteasome. Elimination of RLE-1 expression in C. elegans causes increased transcriptional activation and sustained nuclear localization of DAF-16. Overexpression of DAF-16 in rle-1 mutants increases worm lifespan, while disruption of DAF-16 expression in rle-1 mutants reverses their longevity. Thus, RLE-1 is an E3 ubiquitin ligase of DAF-16 that regulates C. elegans aging.

背景与目标： ：叉头转录因子DAF-16是线虫中胰岛素/ IGF-1信号传导途径的下游靶标，对于寿命调节和抗逆性都是必不可少的。调节DAF-16转录激活所涉及的分子机制仍然不确定。在这里，我们已经确定了一种E3泛素连接酶RLE-1（E3调节寿命），该酶调节秀丽隐杆线虫的衰老。秀丽隐杆线虫中RLE-1表达的破坏增加了寿命；寿命的延长是由于DAF-16蛋白升高，而不是daf-16 mRNA水平的变化。我们还发现RLE-1催化DAF-16泛素化，导致蛋白酶体降解。消除秀丽隐杆线虫中的RLE-1表达会导致DAF-16的转录激活增加和持续的核定位。 DAF-16在rle-1突变体中的过表达延长了蠕虫的寿命，而在rle-1突变体中破坏DAF-16的表达则逆转了它们的寿命。因此，RLE-1是DAF-16的一种E3泛素连接酶，可调节秀丽线虫的衰老。

BACKGROUND & AIMS: :Macrophages are a critically important component of the innate and adaptive immune systems. They are equipped with oxidative and non-oxidative mechanisms to kill ingested pathogens. Natural Killer Lytic-Associated Molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killer cells. We show that NKLAM expression in macrophages was enhanced by Toll-like receptor agonists and pro-inflammatory cytokines. Using confocal microscopy, we found that NKLAM colocalized with ingested Escherichia coli. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAM translocated to the phagosome early during maturation at a time that coincided with elevated levels of ubiquitinated phagosome proteins. In killing assays with bone marrow-derived macrophages from wild type and NKLAM-deficient mice, we found that NKLAM-deficient macrophages demonstrated less killing of E. coli than wild type macrophages. Collectively, our data show that NKLAM is a novel component of macrophage phagosomes and is involved in macrophage bactericidal functions.

背景与目标： 巨噬细胞是先天和适应性免疫系统的至关重要的组成部分。它们配备了氧化和非氧化机制，可以杀死摄入的病原体。天然杀手液相关分子（NKLAM）是在巨噬细胞和天然杀手细胞中表达的E3泛素连接酶。我们显示，巨噬细胞中的NKLAM表达被Toll样受体激动剂和促炎性细胞因子增强。使用共聚焦显微镜，我们发现NKLAM与摄入的大肠杆菌共定位。在使用IgG调理乳胶珠作为靶标的测定中，我们证明了NKLAM在成熟过程中的早期就易位到吞噬体，而此时泛素化的吞噬体蛋白水平升高。在用野生型和NKLAM缺陷型小鼠的骨髓巨噬细胞进行的杀伤试验中，我们发现NKLAM缺陷型巨噬细胞对大肠杆菌的杀灭作用少于野生型巨噬细胞。总体而言，我们的数据表明NKLAM是巨噬细胞吞噬体的一种新型成分，并参与巨噬细胞的杀菌功能。

BACKGROUND & AIMS: The Mg(2+)-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg(2+), ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E.AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP(2)O(7)(2-) and the enzyme.MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme.nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K(d) of Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophilesthe lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.

背景与目标： Mg（2）依赖的T4 DNA和RNA连接酶的腺苷酸化在不存在DNA底物的情况下使用瞬时光吸收和荧光光谱进行了研究。 Mg（2），ATP和焦磷酸盐的浓度发生了系统性的变化，结果得出的结论是，核苷酸基转移是根据两种金属离子机理进行的。根据此机制，只有二价镁配体形式Mg（2）ATP（0）与酶反应形成共价复合物E.AMP。逆反应（ATP合成）发生在单镁配位的焦磷酸盐形式MgP（2）O（7）（2-）与酶MgAMP复合物之间。核苷酸结合率降低顺序ATP（4-）> MgATP（2-）> Mg（2）ATP（0），表明非共价酶与核苷酸复合物的形成是由静电相互作用驱动的。与RNA连接酶相比，T4 DNA连接酶显示出显着更高的ATP结合率和随后的腺苷酸化，部分原因是它使结合酶的Mg（2）ATP（0）的Mg（2）的K（d）降低10以上。 -折叠。为了阐明Mg（2）在T4 DNA和RNA连接酶催化的核苷酸转移中的作用，我们提出了一种过渡态构型，其中Mg（2）催化离子与两个亲核试剂反应形成酶的赖氨酰部分氨基磷酸酯键和ATP的α-β桥接氧。

BACKGROUND & AIMS: :Tagging a small molecule ubiquitin to a protein substrate, or protein ubiquitination, plays an important role in the immune responses. This process is catalyzed by a cascade of enzymatic reactions, with the E3 ubiquitin ligases being the critical enzymes that determine the specificity of substrate recognition. The E3 ligase Itch was identified from a mutant mouse which displays skin scratching and abnormal immune disorders. In the past few years, much progress has been made in our understanding of Itch-promoted protein ubiquitination, modulation of its ligase activity by upstream kinases, and the kinase-ligase interaction in T cell differentiation and tolerance induction.

背景与目标： 在蛋白质底物上标记小分子泛素或蛋白质泛素化在免疫反应中起重要作用。 E3泛素连接酶是决定底物识别特异性的关键酶，可通过级联的酶促反应来催化这一过程。从显示皮肤抓挠和异常免疫异常的突变小鼠中鉴定出E3连接酶瘙痒。在过去的几年中，在我们对Itch促进的蛋白泛素化，上游激酶对其连接酶活性的调节以及T细胞分化和耐受诱导中激酶-连接酶相互作用的理解上取得了很大进展。

BACKGROUND & AIMS: :BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.

背景与目标： ：BRCA1 / 2通过稳定停滞的叉子来帮助维持基因组完整性。在这里，我们将E3连接酶RFWD3确定为BRCA2缺陷细胞失速叉稳定性的重要调节剂，并表明RFWD3的编码缺失可在复制压力下挽救叉的降解，塌陷和细胞敏感性。 BRCA2缺陷细胞中的叉子停滞，积累了磷酸化和泛素化的复制蛋白A（ubq-pRPA），后者是由RFWD3介导的。生成此中间体需要SMARCAL1，这表明它取决于停滞的货叉反转。我们显示，在BRCA2缺失的细胞中，抢救货叉降解可能不足以确保货叉修复。在缺乏BRCA2的细胞中消耗MRE11确实可以阻止前叉降解，但是在存在复制压力的情况下，它不能防止前叉塌陷和细胞敏感性。在缺乏BRCA1的细胞中没有这种ubq-pRPA中间体形成，我们的结果表明BRCA1可能在停滞的叉子修复途径中的BRCA2上游起作用。总体而言，我们的数据揭示了RFWD3破坏BRCA2缺陷细胞中叉子的稳定性的新机制。

BACKGROUND & AIMS: :Recent developments in pulmonary cell biology have shown that the maintenance of protein concentrations, proteostasis, is an integral process of all biologic systems. The balance of available protein is the sum total of three key elements of cell metabolism: production by transcription and translation, compartmentalization through processing and sorting, and proteolytic degradation of proteins at any stage of their life-span. Considerable advances are constantly made in each of these three essential fields, and our appreciation for the diversity of mechanisms of protein degradation has expanded greatly in the last decade. The ubiquitin proteasome system (UPS) has emerged as the predominant protein degradation pathway in eukaryotes, with the large cullin-RING family of E3 ligases responsible for ubiquitination of a broad array of proteins to be degraded. The Skip-Cullin-F-box (SCF) ubiquitin E3 ligase superfamily is the largest family of cullin-RING ligases, with interchangeable F-box proteins orchestrating the trafficking proteins for ubiquitination and degradation. We will discuss the best characterized and most recent developments in the role of this intriguing family of proteins in normal physiology and disorders of the lungs.

背景与目标： ：肺细胞生物学的最新发展表明，蛋白质浓度（蛋白稳态）的维持是所有生物系统不可或缺的过程。可用蛋白质的平衡是细胞代谢的三个关键要素的总和：通过转录和翻译进行生产，通过加工和分类进行区室化以及在蛋白质生命周期的任何阶段进行蛋白水解降解。在这三个必不可少的领域中，每个领域都不断取得重大进展，并且在过去的十年中，我们对蛋白质降解机制多样性的认识大大提高。泛素蛋白酶体系统（UPS）已成为真核生物中主要的蛋白质降解途径，其中大的cullin-RING E3连接酶家族负责多种降解蛋白质的泛素化。 Skip-Cullin-F-box（SCF）泛素E3连接酶超家族是cullin-ring连接酶的最大家族，可互换的F-box蛋白协调运输蛋白的泛素化和降解。我们将讨论这个有趣的蛋白质家族在正常生理和肺部疾病中的作用中最有特色的和最新的发展。

BACKGROUND & AIMS: Protein ubiquitination plays an important role in regulating the abundance and conformation of a broad range of eukaryotic proteins. This process involves a cascade of enzymes including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). E1 and E2 represent two families of structurally related proteins and are relatively well characterized. In contrast, the nature and mechanism of E3, proposed to contain activities in catalyzing isopeptide bond formation (ubiquitin ligation) and substrate targeting, remains inadequately understood. Two major families of E3 ubiquitin ligases, the HECT (for homologous to E6-AP C terminus) family and the RING family, have been identified that utilize distinct mechanisms in promoting isopeptide bond formation. Here, we showed that purified RING finger domain of ROC1, an essential subunit of SKP1-cullin/CDC53-F box protein ubiquitin ligases, was sufficient to activate UBCH5c to synthesize polyubiquitin chains. The sequence flanking the RING finger in ROC1 did not contribute to UBCH5c activation, but was required for binding with CUL1. We demonstrated that all cullins, through their binding with ROC proteins, constituted active ubiquitin ligases, suggesting the existence in vivo of a large number of cullin-RING ubiquitin ligases. These results are consistent with the notion that the RING finger domains allosterically activate E2. We suggest that RING-E2, rather than cullin-RING, constitutes the catalytic core of the ubiquitin ligase and that one major function of the cullin subunit is to assemble the RING-E2 catalytic core and substrates together.

背景与目标： 蛋白质泛素化在调节各种真核蛋白质的丰度和构象中起着重要作用。此过程涉及一连串的酶，包括泛素激活酶（E1），泛素结合酶（E2）和泛素连接酶（E3）。 E1和E2代表结构相关蛋白的两个家族，并且具有相对较好的特征。相比之下，人们对E3的性质和机理提出了建议，其中E3包含催化异肽键形成（泛素连接）和底物靶向的活性，但尚未充分了解。已鉴定出两个主要的E3泛素连接酶家族，即HECT（与E6-AP C端同源）家族和RING家族，它们利用不同的机制促进异肽键的形成。在这里，我们表明，ROC1的纯化RING指结构域是SKP1-cullin / CDC53-F框蛋白泛素连接酶的必需亚基，足以激活UBCH5c以合成聚泛素链。 ROC1的RING指旁的序列没有促进UBCH5c激活，但是与CUL1结合是必需的。我们证明了所有cullins通过与ROC蛋白结合而构成了活性泛素连接酶，表明体内存在大量cullin-RING泛素连接酶。这些结果与RING指结构域变构激活E2的观点一致。我们建议RING-E2而非cullin-RING构成泛素连接酶的催化核心，而cullin亚基的主要功能之一是将RING-E2催化核心和底物组装在一起。

BACKGROUND & AIMS: :Loss of function of the tumor suppressor protein BRCA1 is responsible for a high percentage of familial and also sporadic breast cancers. Early work identified a stimulatory transcriptional coactivator function for the BRCA1 protein, and more recently, BRCA1 has been implicated in transcriptional repression, although few examples of repressed genes have been characterized. We recently used an in vitro transcription assay to identify a biochemical mechanism that explained the BRCA1 stimulatory activity. In this study, we identified an ubiquitin-dependent mechanism by which BRCA1 inhibits transcription. BRCA1 ubiquitinates the transcriptional preinitiation complex, preventing stable association of TFIIE and TFIIH, and thus blocks the initiation of mRNA synthesis. What is striking about this mechanism of regulation by BRCA1 is that the ubiquitination of the preinitiation complex is not targeting proteins for degradation by the proteasome, nor are ubiquitin receptors modifying the activity, but rather the ubiquitin moiety itself interferes with the assembly of basal transcription factors at the promoter. Using RNAi to knockdown expression of the endogenous BRCA1 protein, we assessed the level of repression dependent on BRCA1 in the cell, and we found that BRCA1 is at least as significant a transcriptional repressor as it is an activator. These results define a biochemical mechanism by which the BRCA1 enzymatic activity regulates a key cellular process.

背景与目标： ：抑癌蛋白BRCA1的功能丧失导致家族性和散发性乳腺癌的发生率很高。早期的工作确定了BRCA1蛋白具有刺激性的转录共激活因子功能，最近，BRCA1与转录阻抑有关，尽管已鉴定出一些抑制基因的例子。我们最近使用了体外转录测定法来鉴定解释BRCA1刺激活性的生化机制。在这项研究中，我们确定了BRCA1抑制转录的泛素依赖性机制。 BRCA1泛素化转录预启动复合物，阻止TFIIE和TFIIH的稳定缔合，从而阻止mRNA合成的启动。 BRCA1调控机制的惊人之处在于，预启动复合物的泛素化作用既不是针对蛋白酶体降解的蛋白质，也不是泛素受体改变了活性，而是泛素部分本身干扰了基础转录因子的组装。在启动子上。使用RNAi敲低内源性BRCA1蛋白的表达，我们评估了依赖于细胞中BRCA1的抑制水平，我们发现BRCA1至少与激活因子一样重要。这些结果定义了一种生物化学机制，BRCA1酶活性通过该生物化学机制调节关键的细胞过程。