Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase

The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.     

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity

Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.     

U-box蛋白质的结构及其功能研究进展

泛素系统是选择性降解细胞内蛋白质的重要系统之一,U-box蛋白质是此系统中决定底物特异性识别的一种新型E3蛋白质,部分U-box蛋白质属于泛素链聚集因子-E4。U-box结构域大约由70个氨基酸残基构成,在酵母、植物和动物等真核生物中保守存在,但植物中的数目远多于动物中。该蛋白质在细胞内异常蛋白质的降解及质量控制方面具有重要的作用,了解U-box蛋白质的功能对疾病的发生控制机理有重要意义。     

两个高度同源的RING结构域蛋白功能初步研究

泛素化途径在植物应答生物和非生物胁迫中起着重要作用,其中E3连接酶在泛素化途径中起着决定性作用。室通过在线基因芯片预测发现2个与非生物胁迫相关的拟南芥RING结构域蛋白基因At2g24480（HHR1）、At5g43200(HHR2)通过体外泛素化实验证明了二者具有E3连接酶活性。对这两个E3连接酶进行了亚细胞定位,表达谱和组织特异性分析,发现二者受诱导后的表达情况、组织特异性、亚细胞定位均不同。HHR1定位于细胞膜上,HHR2定位于细胞核;表达谱分析表明HHR1响应热和H2O2胁迫,而HHR2响应盐和H2O2胁迫;HHR1在根和花中的表达量较高,在茎和叶中的表达量较低,而HHR2在根、茎、叶中的表达量都较高,在花中表达量较低。该研究结果证实了HHR1和HHR2的一些性质和亚细胞定位,为深入研究其功能奠定了基础。     

番茄RBX1基因的功能初探

E3泛素连接酶是泛素蛋白酶体途径中非常重要的蛋白复合体,而大部分的E3连接酶都以Cullin蛋白作为支架,所有基于Cullin蛋白的 E3 连接酶都含有一个催化亚基RBX1。RBX1对于E3复合体结合E2和Cullin蛋白的活化具有重要作用。利用酵母双杂交技术鉴定出番茄RBX1蛋白与番茄CUL4蛋白有直接的相互作用。通过荧光定量PCR（Real-time PCR）技术分析野生型番茄中RBX1基因的组织特异性,结果显示RBX1基因在番茄各组织中均有表达,但在花与果实中表达量较高,茎和叶中相对较少。同时构建植物过量表达载体 PBI121-35S-RBX1并转化番茄,获得 3 株过量表达和 3 株RBX1共抑制转基因植株。研究发现共抑制的转基因植株表现出叶片变大、顶端优势变弱和不育等表型,说明RBX1基因在番茄的生长发育中发挥重要作用,推测植物RBX1基因可能与生长素和油菜素内酯等植物激素应答以及细胞增殖有关,为进一步研究该基因在植物中的生物学功能提供了线索。     

S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.     

RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites

Mutations affecting the BRCT domains of the breast cancer-associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.     

ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser(387) and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser(387) was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser(387) was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.     

Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein

Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion.     

Antigen presentation requires transport of MHC class I molecules from the endoplasmic reticulum

The role of exocytosis of major histocompatibility complex (MHC) class I molecules in the presentation of antigens to mouse cytotoxic T lymphocytes (CTLs) was examined by use of a recombinant vaccinia virus that expresses the E19 glycoprotein from adenovirus. E19 blocked the presentation of vaccinia and influenza virus proteins to CTLs in a MHC class I allele-specific manner identical to its inhibition of MHC class I transport from the endoplasmic reticulum. This finding indicates that (i) the relevant parameter for antigen presentation is the rate of MHC class I molecule exocytosis, not the level of class I cell surface expression, and (ii) association of class I molecules with antigen is likely to occur within the endoplasmic reticulum.     

Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli

To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.     

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.     

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase

Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.     

Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.     

TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.     

Coordinated nonvectorial folding in a newly synthesized multidomain protein

The low-density lipoprotein receptor (LDL-R) is a typical example of a multidomain protein, for which in vivo folding is assumed to occur vectorially from the amino terminus to the carboxyl terminus. Using a pulse-chase approach in intact cells, we found instead that newly synthesized LDL-R molecules folded by way of "collapsed" intermediates that contained non-native disulfide bonds between distant cysteines. The most amino-terminal domain acquired its native conformation late in folding instead of during synthesis. Thus, productive LDL-R folding in a cell is not vectorial but is mostly posttranslational, and involves transient long-range non-native disulfide bonds that are isomerized into native short-range cysteine pairs.