• 【Praja1 E3泛素连接酶通过p38α激活后EZH2的降解促进骨骼肌发生。】 复制标题 收藏 收藏
    DOI:10.1038/ncomms13956 复制DOI
    作者列表:Consalvi S,Brancaccio A,Dall'Agnese A,Puri PL,Palacios D
    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。
  • 【新型RING E3泛素连接酶在乳腺癌中的作用。】 复制标题 收藏 收藏
    DOI:10.1593/neo.06469 复制DOI
    作者列表:Burger A,Amemiya Y,Kitching R,Seth AK
    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。
  • 【番茄对豆类病原体丁香假单胞菌PV的非寄主抗性。丁香香蒲B728a是由于avrptobb728a中的E3泛素连接酶结构域有缺陷。】 复制标题 收藏 收藏
    DOI:10.1094/MPMI-08-12-0190-R 复制DOI
    作者列表:Chien CF,Mathieu J,Hsu CH,Boyle P,Martin GB,Lin NC
    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。
  • 【RLE-1,一种E3泛素连接酶,通过催化DAF-16多聚泛素化来调节线虫的衰老。】 复制标题 收藏 收藏
    DOI:10.1016/j.devcel.2006.12.002 复制DOI
    作者列表:Li W,Gao B,Lee SM,Bennett K,Fang D
    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。
  • 【E3泛素连接酶NKLAM是巨噬细胞吞噬体蛋白,在细菌杀灭中起作用。】 复制标题 收藏 收藏
    DOI:10.1016/j.cellimm.2012.09.004 复制DOI
    作者列表:Lawrence DW,Kornbluth J
    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。
  • 【T4 DNA和RNA连接酶催化的Mg2依赖性核苷酸转移的动力学机理。】 复制标题 收藏 收藏
    DOI:10.1074/jbc.M109616200 复制DOI
    作者列表:Cherepanov AV,de Vries S
    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。

  • 【E3泛素连接酶Itch在T细胞的活化,分化和耐受性方面。】 复制标题 收藏 收藏
    DOI:10.1016/j.smim.2007.02.003 复制DOI
    作者列表:Liu YC
    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。
  • 【E3连接酶RFWD3是BRCA2缺陷型细胞中失速叉稳定性的新型调节剂。】 复制标题 收藏 收藏
    DOI:10.1083/jcb.201908192 复制DOI
    作者列表:Duan H,Mansour S,Reed R,Gillis MK,Parent B,Liu B,Sztupinszki Z,Birkbak N,Szallasi Z,Elia AEH,Garber JE,Pathania S
    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。
  • 【关于肺中SCF泛素E3连接酶功能的新见解。】 复制标题 收藏 收藏
    DOI:10.1016/j.cellsig.2013.05.003 复制DOI
    作者列表:Weathington NM,Mallampalli RK
    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。
  • 【ROC1的RING指激活UBC5泛素结合酶,所有cullins组装活性泛素连接酶。】 复制标题 收藏 收藏
    DOI:10.1074/jbc.M108565200 复制DOI
    作者列表:Furukawa M,Ohta T,Xiong Y
    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。

  • 【依赖于BRCA1 E3泛素连接酶的转录抑制机制。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.0610481104 复制DOI
    作者列表:Horwitz AA,Affar el B,Heine GF,Shi Y,Parvin JD
    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。
  • 【小分子TSC01682通过特异性破坏CUL4B-DDB1相互作用并减少CRL4B E3连接酶底物的泛素化来抑制骨肉瘤细胞的生长。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Chen B,Feng Y,Zhang M,Cheng G,Chen B,Wang H
    BACKGROUND & AIMS: :The direct interaction between Cullin 4B (CUL4B) and DNA damage-binding protein 1 (DDB1) is required for the assembly of Cullin4B-RING E3 ligase complex (CRL4B), which are involved in the tumorigenesis of osteosarcoma through ubiquitinating and degrading multiple tumor suppressors and cell cycle regulators. Thus, targeting CUL4B-DDB1 interaction to prevent the assembly of CRL4B may be a potent approach to inhibit osteosarcoma cell growth. In the present study, we identified six naturally-sourced small molecules that can specifically disrupt the CUL4B-DDB1 interaction using an in vitro high-throughput screening (HTS) system in yeast. We focused our investigation on revealing the molecular effects of TSC01682, the most active compound capable of inhibiting osteosarcoma cell growth. Biochemically, TSC01682 significantly repressed the CUL4B-DDB1 interaction in both yeast cells and osteosarcoma cells. Moreover, TSC01682 treatment in osteosarcoma cells also caused a decrease of other CRL4B components including CUL4-associated factor 11 (DCAF11) and DCAF13, but an increase of two CRL4B substrates including cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through inhibiting their ubiquitination. Consistent with these molecular changes, TSC01682 treatment significantly inhibited cell proliferation, colony formation, invasion, and in vivo tumor growth. Collectively, our results suggest that TSC01682 is a potent compound capable of disrupting the CUL4B-DDB1 interaction, and it may be developed as a chemotherapeutic drug for osteosarcoma treatment.
    背景与目标: :Cullin4B-RING E3连接酶复合物(CRL4B)的组装需要Cullin 4B(CUL4B)和DNA损伤结合蛋白1(DDB1)之间的直接相互作用,而Cullin4B-RING E3连接酶复合物(CRL4B)通过泛素化和降解多个肿瘤参与骨肉瘤的肿瘤发生抑制剂和细胞周期调节剂。因此,靶向CUL4B-DDB1相互作用以防止CRL4B的组装可能是抑制骨肉瘤细胞生长的有效方法。在本研究中,我们鉴定了六个天然来源的小分子,它们可以使用酵母中的体外高通量筛选(HTS)系统特异性破坏CUL4B-DDB1的相互作用。我们的研究集中在揭示TSC01682的分子作用,TSC01682是能够抑制骨肉瘤细胞生长的最具活性的化合物。生化方面,TSC01682显着抑制酵母细胞和骨肉瘤细胞中的CUL4B-DDB1相互作用。此外,在骨肉瘤细胞中进行TSC01682处理还导致其他CRL4B组分(包括CUL4相关因子11(DCAF11)和DCAF13)减少,但增加了两种CRL4B底物,包括细胞周期蛋白依赖性激酶抑制剂1A(CDKN1A,也称为p21)和磷酸酶和张力蛋白同源物通过抑制它们的泛素化作用而在第10号染色体(PTEN)上缺失。与这些分子变化一致,TSC01682处理可显着抑制细胞增殖,集落形成,侵袭和体内肿瘤生长。总的来说,我们的结果表明,TSC01682是一种能够破坏CUL4B-DDB1相互作用的有效化合物,它可能被开发为用于骨肉瘤治疗的化疗药物。
  • 【低水平的DNA连接酶III和IV足以产生有效的NHEJ。】 复制标题 收藏 收藏
    DOI:10.1002/jcp.21120 复制DOI
    作者列表:Windhofer F,Wu W,Iliakis G
    BACKGROUND & AIMS: :Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.
    背景与目标: :高等真核生物细胞主要通过非同源DNA末端连接(NHEJ)途径重新结合其DNA中的双链断裂(DSB),该途径也利用DNA-PKcs,Ku,LIG4,XRCC4,XLF / Cernunnos,Artemis的产物作为DNA聚合酶lambda(称为D-NHEJ)。这些蛋白质中有缺陷的突变体利用NHEJ的备用途径(B-NHEJ)从其基因组中去除了大部分DSB。虽然D-NHEJ仅依赖于DNA连接酶IV,但最近的工作指出DNA连接酶III是B-NHEJ的组成部分。在这里,我们使用RNA干扰(RNAi)来进一步研究NHEJ途径中DNA连接酶III和IV的活性要求。我们报告说,在D-NHEJ熟练的细胞中,或在D-NHEJ受到化学或遗传损害的细胞中,LIG3表达的70-80%的降低对DSB重新结合没有可检测的作用。出人意料的是,敲低LIG4对修复熟练的细胞也没有影响,但会抑制DSB在放射敏感性细胞系中重新结合,并导致亚型LIG4突变严重损害其活性。结果表明,通过极低的连接酶水平可完全覆盖D-NHEJ或B-NHEJ,并证明具有LIG4突变的患者细胞中DNA连接酶IV的残留末端连接。
  • 【泛素蛋白连接酶-新的治疗靶标?】 复制标题 收藏 收藏
    DOI:10.2174/1389203043379800 复制DOI
    作者列表:Robinson PA,Ardley HC
    BACKGROUND & AIMS: :Intracellular protein degradation is a tightly regulated process that in many cases is controlled by protein ubiquitylation. The ubiquitin pathway is a major route by which cells not only remove normal proteins at the appropriate time but also abnormally folded normal or mutant, cytoplasmic and membrane, proteins. This has led to a major impetus to identify constituents of the pathway. The key components that regulate substrate ubiquitylation are the ubiquitin-protein ligases. Ligases come in many forms, from single proteins to very large multiprotein complexes. Specificity of targeting can be modulated by the requirement for post-translational modifications such as phosphorylation, hydroxylation or oxidation of the substrate and, in some cases, the ligase itself. The requirement for substrate modification prior to ubiquitylation allows the same ligase to target different substrates within the same cell at different times. Abnormal intracellular protein processing is a common feature of many human diseases including neurodegenerative diseases and cancer. It may not represent the causative factor that initiates the disease process but may be a downstream regulator of the toxic effect. These abnormalities often arise from the loss of a key protein-protein interaction. As a consequence, mutated proteins can have very different half-lives from their normal counterparts. This can affect the levels of their activity and/or lead to the formation of protein aggregates (inclusion bodies/aggresomes). In this review, we aim to highlight examples of diseases where abnormal protein ubiquitylation is proposed to be a key regulator of the disease process. The recent success of the proteasome inhibitor Bortezomib (PS-341) for treatment of relapsed, refractory myeloma suggests that the modulation of individual ubiquitin-protein ligase activities with synthetic agents may represent a novel approach that has enormous potential for the treatment of a wide range of diseases.
    背景与目标: :细胞内蛋白质降解是一个严格调控的过程,在许多情况下是由蛋白质泛素化控制的。遍在蛋白途径是细胞不仅在适当的时间去除正常蛋白而且在正常或突变的细胞质和膜蛋白中异常折叠的主要途径。这导致了确定该途径成分的主要动力。调节底物泛素化的关键成分是泛素蛋白连接酶。从单个蛋白到非常大的多蛋白复合物,甘氨酸有多种形式。靶向的特异性可以通过翻译后修饰的要求来调节,例如底物的磷酸化,羟基化或氧化,在某些情况下还可以是连接酶本身。在泛素化之前对底物进行修饰的要求允许相同的连接酶在不同时间靶向同一细胞内的不同底物。细胞内蛋白质加工异常是许多人类疾病(包括神经退行性疾病和癌症)的普遍特征。它可能不代表引发疾病过程的病因,但可能是毒性作用的下游调节剂。这些异常通常是由于关键蛋白质与蛋白质相互作用的丧失而引起的。结果,突变的蛋白质的半衰期可能与其正常的半衰期截然不同。这可能会影响其活性水平和/或导致蛋白质聚集体(包涵体/聚集体)的形成。在这篇综述中,我们旨在突出疾病的例子,其中异常蛋白质泛素化被认为是疾病过程的关键调节因子。蛋白酶体抑制剂Bortezomib(PS-341)在治疗复发性难治性骨髓瘤方面的最新成功表明,合成剂对单个泛素蛋白连接酶活性的调节可能代表了一种新颖的方法,具有广泛的治疗潜力。疾病。
  • 【一对E3泛素连接酶竞争调节子弹动力学和轴突指导。】 复制标题 收藏 收藏
    DOI:10.1083/jcb.201902088 复制DOI
    作者列表:Boyer NP,McCormick LE,Menon S,Urbina FL,Gupton SL
    BACKGROUND & AIMS: :Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the filopodial actin polymerase VASP and consequently filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1-dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, filopodial stability, and axon guidance.
    背景与目标: :适当的轴突引导对于形成准确的神经元连接是必要的。刺激生长锥内细胞骨架重组的轴突引导提示直接轴突导航。长期以来,生长锥周围的丝足病一直被认为是轴突引导线索的传感器,但是它们对细胞外线索的反应方式仍然不清楚。我们以前的工作发现,通过非降解的TRIM9依赖性泛素化作用,对腓肠肌肌动蛋白聚合酶VASP以及由此引起的腓肠肌稳定性进行了负调控。轴突转动需要适当的VASP泛素化和去泛素化以响应netue-1指导。在这里,我们显示TRIM9相关蛋白TRIM67胜过TRIM9与VASP的相互作用,并拮抗TRIM9依赖的VASP泛素化。 netrin-1依赖的丝状副反应,轴突转向和分支以及纤维束形成需要两种紧密相关的E3泛素连接酶的令人惊讶的拮抗作用。我们建议一种新颖的模型,其中通过一对干扰的连接酶对VASP泛素化的协调调节是VASP动力学,副手稳定性和轴突导向的关键要素。

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