Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue

We previously showed that Cidea(-/-) mice are resistant to diet-induced obesity through the upregulation of energy expenditure. The AMP-activated protein kinase (AMPK), consisting of catalytic alpha subunit and regulatory subunits beta and gamma, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea(-/-) mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the beta subunit of AMPK, but not with the alpha or gamma subunit. When co-expressed with Cidea, the stability of AMPK-beta subunit was dramatically reduced due to increased ubiquitination-mediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea(-/-) adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea-mediated ubiquitin-dependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea-null mice.     

PHF23 (plant homeodomain finger protein 23) negatively regulates cell autophagy by promoting ubiquitination and degradation of E3 ligase LRSAM1

Autophagy is a multistep process that involves the degradation and digestion of intracellular components by the lysosome. It has been proved that many core autophagy-related molecules participate in this event. However, new component proteins that regulate autophagy are still being discovered. At present, we report PHF23 (PHD finger protein 23) with a PHD-like zinc finger domain that can negatively regulate autophagy. Data from experiments indicated that the overexpression of PHF23 impaired autophagy, as characterized by decreased levels of LC3B-II and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, knockdown of PHF23 resulted in opposite effects. Molecular mechanism studies suggested that PHF23 interacts with LRSAM1, which is an E3 ligase key for ubiquitin-dependent autophagy against invading bacteria. PHF23 promotes the ubiquitination and proteasome degradation of LRSAM1. We also show that the PHD finger of PHF23 is a functional domain needed for the interaction with LRSAM1. Altogether, our results indicate that PHF23 is a negative regulator associated in autophagy via the LRSAM1 signaling pathway. The physical and functional connection between the PHF23 and LRSAM1 needs further investigation.     

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.     

Synoviolin promotes IRE1 ubiquitination and degradation in synovial fibroblasts from mice with collagen-induced arthritis

The E3 ubiquitin ligase synoviolin (SYVN1) functions as an anti-apoptotic factor that is responsible for the outgrowth of synovial cells during the development of rheumatoid arthritis. The molecular mechanisms underlying SYVN1 regulation of cell death are largely unknown. Here, we report that elevated SYVN1 expression correlates with decreased levels of the protein inositol-requiring enzyme 1 (IRE1)-a pro-apoptotic factor in the endoplasmic reticulum (ER)-stress-induced apoptosis pathway-in synovial fibroblasts from mice with collagen-induced arthritis (CIA). SYVN1 interacts with and catalyses IRE1 ubiquitination and consequently promotes IRE1 degradation. Suppression of SYVN1 expression in synovial fibroblasts from CIA mice restores IRE1 protein expression and reverses the resistance of ER-stress-induced apoptosis of CIA synovial fibroblasts. These results show that SYVN1 causes the overgrowth of synovial cells by degrading IRE1, and therefore antagonizes ER-stress-induced cell death.     

Coupled deglycosylation-ubiquitination cascade in regulating PD-1 degradation by MDM2

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Nrdp1S,short variant of Nrdp1, inhibits human glioma progression by increasing Nrdp1‐mediated ErbB3 ubiquitination and degradation

The ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp1) is involved in the induction of apoptosis and suppression of tumour formation. We previously showed that it was expressed at lower levels in human glioma tissues compared with normal brain tissues. However, the mechanism underlying this is unclear. Here, we reported that a novel short variant (Nrdp1S), lacking 71 amino acids at the N‐terminal, was expressed in normal human brain tissue, but absent from glioma tissues. Similar to Nrdp1, Nrdp1S could be degraded by the proteasomal pathway, but exhibited an even longer half‐life than Nrdp1. Nrdp1S was also shown to form a heterodimer with Nrdp1, which increased its stability, thereby augmenting the Nrdp1‐mediated ubiquitination and degradation of ErbB3. EdU incorporation, MTT assay and in vitro colony formation demonstrated that Nrdp1S significantly inhibited the cell tumourigenicity. These results together suggest that Nrdp1S is a tumour suppressor that which potentiates the Nrdp1‐mediated ubiquitination and degradation of ErbB3. An Nrdp1S deficiency may also be an important factor in the loss of Nrdp1.     

CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation

The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.     

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5p_C6A, Pex5p_C6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5p_C6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5p_C6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.     

嗜肺军团菌调节宿主泛素化途径的效应因子及其分子机制研究进展

嗜肺军团菌是一种胞内寄生菌,其通过特有的Dot/IcmType-IVB分泌系统向胞浆内分泌大量效应因子,其中已知参与宿主泛素化调控的效应因子有十多种.这些效应因子通过对宿主泛素化途径进行调控来达到逃避宿主免疫系统"监视"并大量增殖的目的.参与调控宿主泛素化途径的效应因子包括 AnkB、SidC、LubX、SidH、Le...     

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate‐specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase–substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well‐known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene‐silencing suppressor p19 and by time‐courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

BTB蛋白泛素化介导植物发育和逆境应答的研究进展

BTB (broad-complex, tramtrack, and bric-à-brac)结构域是在真核生物中发现的高度保守的蛋白质相互作用基序。含有BTB结构域的一类蛋白统称为BTB蛋白,它们广泛参与转录调控、蛋白质降解等过程。越来越多的研究表明,该基因在植物生长发育、生物与非生物胁迫等生理过程中具有重要的作用。本文以蛋白结构域为基础,系统总结了该基因家族蛋白在泛素化介导植物发育和逆境应答等过程中的研究进展,为植物中该类基因的研究提供了参考。     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.