Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.     

Backbone and side chain 1H, 13C, and 15N assignments of the ubiquitin-like domain of human HOIL-1L, an essential component of linear ubiquitin chain assembly complex

HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date. Here we report backbone and side chain NMR assignments of the UBL of human HOIL-1L. By inspection of chemical shift index, it was predicted that HOIL-1L-UBL assumes a Ub fold followed by an α-helical segment, offering the basis for determination its 3D structure and interaction with HOIP-UBA in solution.     

Biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (LUBAC)

Linear ubiquitylation, in which ubiquitin units are covalently linked through N- and C-terminal amino acids, is a unique cellular signaling mechanism. This process is controlled by a single E3 ubiquitin ligase, the linear ubiquitin chain assembly complex (LUBAC), which is composed of three proteins – HOIL-1L, HOIP and SHARPIN. LUBAC is involved in the activation of the canonical NF-κB pathway and has been linked to NF-κB dependent malignancies. In this work, we present HOIP-based stapled alpha-helical peptides designed to inhibit LUBAC through the disruption of the HOIL-1L-HOIP interaction and loss of the functional complex. We find our HOIP peptides to be active LUBAC ubiquitylation inhibitors in vitro, though through interaction with HOIP rather than HOIL. Active peptides were shown to have inhibitory effects on cell viability, reduced NF-κB activity and decreased production of NF-κB related gene products. This work further demonstrates the potential of LUBAC as a therapeutic target and of the use of stapled peptides as inhibitors of protein-protein interactions.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.     

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear'' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain'' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains.     

Linear Ubiquitination of NEMO Negatively Regulates the Interferon Antiviral Response through Disruption of the MAVS-TRAF3 Complex

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

An ubiquitin ligase recognizing a protein oxidized by iron: implications for the turnover of oxidatively damaged proteins

Protein oxidation is a natural consequence of aerobic metabolism in cells. Oxidative modification of amino acid residues of proteins causes to lose activity or function of proteins. Organisms have thus developed pathways to remove oxidized proteins by rapid protein degradation. These pathways are important components in cellular quality control mechanisms. It has been suggested that oxidized proteins are degraded by the proteasome. However, whether ubiquitylation is necessary for the degradation of oxidized proteins remains a controversial issue. We have recently identified HOIL-1 (heme-oxidized IRP2 ubiquitin ligase-1) as an E3 ligase that recognizes a protein that has been oxidized by iron. This review describes the recent progress made in understanding the ubiquitin-proteolytic pathway and the regulation of iron metabolism. The process involved in eliminating oxidized proteins and the possible roles that HOIL-1 ubiquitin ligase may play in these processes are discussed.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Phosphoinositide-dependent kinase phosphorylation of protein kinase C Apl II increases during intermediate facilitation in aplysia

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine mollusk Aplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation.     

Receptor for activated C kinase (RACK) and protein kinase C (PKC) in egg activation

In somatic cells, translocation of PKCs is facilitated by receptor for activated C kinase (RACK); however its involvement in egg activation is still elusive. We have followed the translocation pattern of conventional and novel PKCs (cPKCs and nPKCs, respectively) upon egg activation. Confocal microscopy indicated the expression and localization of RACK1, a specific receptor protein for cPKCs. Activation of MII eggs, led to translocation to the egg cortex of PKCα, βII and δ and the co-translocation of RACK1, with both PKCα and PKCβII. The association of PKC and actin, both known to be involved in cortical granules exocytosis (CGE) with RACK1, was demonstrated by co-immunoprecipitation. Egg activation resulted in an increased RACK1 level along with a decreased level of PKCβII. Based on these results, we suggest that upon egg activation, RACK1 shuttles activated cPKCs to the egg cortex, thus facilitating CGE.     

UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development,Linking Linear Ubiquitination to Multiple Autoimmune Diseases

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development.     

植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

Protein kinase C (PKC) isoforms in Drosophila

Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.     

The timing and extent of activation of diacylglycerol-responsive protein kinase-cs determines their ability to inhibit or promote platelet-derived growth factor-dependent DNA synthesis

In response to natural agonists, such as platelet-derived growth factor (PDGF), diacylglycerol-responsive protein kinase Cs (PKCs) are activated at two distinct times, early and mid G1, and only the late activity is required for the transition into S phase. Surprisingly, the potent PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits DNA synthesis when it is added in mid G1. Here we investigated why different PKC agonists had opposing effects on cell proliferation. We found that the magnitude and timing of PKC activation determined their ability to suppress DNA synthesis. Furthermore, potent activation of PKCs resulted in robust Erk activation and elevation of p21(CIP1). Finally, PMA was unable to block PDGF-dependent cell cycle progression in cells that lack p21(CIP1). These findings indicate that only potent activators of PKC were capable of blocking cell cycle progression, and the mechanism appears to involve an elevation of p21(CIP1).