Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase

Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.     

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.     

Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.     

Identification of SCF ubiquitin ligase substrates by global protein stability profiling

Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1-cullin-F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle-stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.     

Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase

Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)-dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.     

Spatial regulation of an E3 ubiquitin ligase directs selective synapse elimination

Stereotyped synaptic connectivity can arise both by precise recognition between appropriate partners during synaptogenesis and by selective synapse elimination. The molecular mechanisms that underlie selective synapse removal are largely unknown. We found that stereotyped developmental elimination of synapses in the Caenorhabditis elegans hermaphrodite-specific motor neuron (HSNL) was mediated by an E3 ubiquitin ligase, a Skp1-cullin-F-box (SCF) complex composed of SKR-1 and the F-box protein SEL-10. SYG-1, a synaptic adhesion molecule, bound to SKR-1 and inhibited assembly of the SCF complex, thereby protecting nearby synapses. Thus, subcellular regulation of ubiquitin-mediated protein degradation contributes to precise synaptic connectivity through selective synapse elimination.     

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.     

Identification of p53 as a sequence-specific DNA-binding protein

The tumor-suppressor gene p53 is altered by missense mutation in numerous human malignancies. However, the biochemical properties of p53 and the effect of mutation on these properties are unclear. A human DNA sequence was identified that binds specifically to wild-type human p53 protein in vitro. As few as 33 base pairs were sufficient to confer specific binding. Certain guanines within this 33-base pair region were critical, as methylation of these guanines or their substitution with thymine-abrogated binding. Human p53 proteins containing either of two missense mutations commonly found in human tumors were unable to bind significantly to this sequence. These data suggest that a function of p53 may be mediated by its ability to bind to specific DNA sequences in the human genome, and that this activity is altered by mutations that occur in human tumors.     

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.     

People with absolute pitch process tones without producing a p300

The P300 is a positive-going component of the event-related potential. In subjects with absolute, or "perfect," pitch, the P300 elicited by the less frequent of two auditory probes is small or absent. In these subjects, visual probes elicit a normal P300. These results support the view of P300 as a manifestation of the updating of working memory.     

Suppression of human colorectal carcinoma cell growth by wild-type p53

Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.     

Genetic mechanisms of tumor suppression by the human p53 gene

Mutations of the gene encoding p53, a 53-kilodalton cellular protein, are found frequently in human tumor cells, suggesting a crucial role for this gene in human oncogenesis. To model the stepwise mutation or loss of both p53 alleles during tumorigenesis, a human osteosarcoma cell line, Saos-2, was used that completely lacked endogenous p53. Single copies of exogenous p53 genes were then introduced by infecting cells with recombinant retroviruses containing either point-mutated or wild-type versions of the p53 cDNA sequence. Expression of wild-type p53 suppressed the neoplastic phenotype of Saos-2 cells, whereas expression of mutated p53 conferred a limited growth advantage to cells in the absence of wild-type p53. Wild-type p53 was phenotypically dominant to mutated p53 in a two-allele configuration. These results suggest that, as with the retinoblastoma gene, mutation of both alleles of the p53 gene is essential for its role in oncogenesis.     

Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.     

小鼠p300-HAT真核表达质粒的构建

[目的]构建小鼠p300-HAT的真核表达质粒。[方法]利用PCR技术扩增p300的HAT域,然后构建p300的HAT域的真核表达质粒（p300-HAT—EGFP）,并通过PCR验证和酶切验证重组质粒的正确性;提取不舍内毒素的质粒,转染HepG2细胞,通过观测绿色荧光检测转染效率。[结果]PcR产物的电泳结果显示,成功扩增了小鼠p300基因HAT结构域,构建了小鼠p300基因HAT结构域的真核表达质粒,将其命名为m-p300-HAT-pEGFP—C1。重组质粒可以成功表达融合绿色荧光蛋白目的基因蛋白。[结论]p300-HAT—EGFP重组质粒的成功构建为后续开展巨噬细胞NF—kB信号通路的研究奠定了基础。