Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.     

泛素在冠心病患者外周血炎性细胞中的表达

目的通过检测冠心病患者外周血炎性细胞泛素表达水平,评价泛素与冠心病的相关性。方法选择经冠状动脉造影确诊的冠心病患者120例,男性86例,女性34例,平均年龄(60.4±7.8)岁。按临床表现分为稳定性心绞痛组(SAP组)、不稳定性心绞痛组(UAP组)、急性心肌梗死组(AMI组),每组40例。同期选择经冠状动脉造影排除冠心病的40例患者为对照组。用RT-PCR检测法及Western blot方法检测4组患者外周血单核细胞和淋巴细胞泛素的mRNA和蛋白表达水平。结果冠心病各组患者外周血泛素mRNA和蛋白表达水平均明显高于对照组(P<0.05),以对照组泛素的mRNA和蛋白表达水平为参照,SAP组、UAP组、AMI组泛素的mRNA和蛋白表达分别为对照组的1.3、1.4倍;1.8、1.6倍;2.2、1.9倍,随病情加重泛素表达量进一步增加。结论泛素可能在冠心病发生、发展中起重要作用。     

Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1

Repeated injection of α-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-γ production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b^−/− and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFκB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1^−/− NKT cells are defective in IFN-γ production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFκB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.     

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway,and its role in yolk sac vascular development and autophagy

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.     

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

Protein–protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.     

Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.     

PROTAC technology: A new drug design for chemical biology with many challenges in drug discovery

Target Protein Degradation TPD is a new avenue and revolutionary for therapeutics because redefining the principles of classical drug discovery and guided by event-based target activity rather than the occupancy-driven activity. Since the discovery of the first PROTAC in 2001, TPD represents a rapidly growing technology, with applications in both drug discovery and chemical biology. Over the last decade, many questions have been raised and today the knowledge gained by each team has elucidated a number of them, although there is still a long way to go. The objective of this work is to present the challenges that the PROTAC strategy has very recently addressed in drug design and discovery by presenting extremely recent results from the literature and to provide guidelines in the drug design of new PROTACs as successful therapeutic modality for medicinal chemists.