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.     

The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.     

TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88

TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.     

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.     

Nedd4-2 ubiquitin ligase interacts with RCAN1 (DSCR1) for the proteasomal degradation

Regulator of Calcineurin 1 (RCAN1/DSCR1/Adapt78) gene is located in the Down syndrome (DS) region of chromosome 21, and critical for the phenotype of DS and Alzheimer disease (AD). In this report, we found that expression of Nedd4-2 E3 ubiquitin ligase decreased the protein level of RCAN1. Decrease of RCAN1 protein expression by Nedd4-2 was blocked by proteasome inhibitor MG132, indicating that this decrease was mediated by the ubiquitin-proteasome pathway. Furthermore, we found that the ability of Nedd4-2 to degrade RCAN1 depended on the direct binding with RCAN1. Consistently, Nedd4-2 enhanced the ubiquitination of RCAN1 protein. Our data provide the first evidence that Nedd4-2 acts as an important regulatory component in the control of RCAN1 protein stability.     

p250GAP Is a Novel Player in the Cdh1-APC/Smurf1 Pathway of Axon Growth Regulation

Axon growth is an essential process during brain development. The E3 ubiquitin ligase Cdh1-APC has emerged as a critical regulator of intrinsic axon growth control. Here, we identified the RhoGAP p250GAP as a novel interactor of the E3 ubiquitin ligase Cdh1-APC and found that p250GAP promotes axon growth downstream of Cdh1-APC. We also report that p250GAP undergoes non-proteolytic ubiquitination and associates with the Cdh1 substrate Smurf1 to synergistically regulate axon growth. Finally, we found that in vivo knockdown of p250GAP in the developing cerebellar cortex results in impaired migration and axonal growth. Taken together, our data indicate that Cdh1-APC together with the RhoA regulators p250GAP and Smurf1 controls axon growth in the mammalian brain.     

CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1

The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.     

SIAH-1 interacts with the Kaposi's sarcoma-associated herpesvirus-encoded ORF45 protein and promotes its ubiquitylation and proteasomal degradation

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

Regulation of the Polycomb protein RING1B ubiquitination by USP7

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.     

E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout C. elegans development

Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB,a virulence effector protein secreted by Salmonella typhimurium

The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonellatyphimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6^−/− mouse embryonic fibroblasts (MEFs) compared with Traf6^+/+ MEFs. However, the ectopic expression of TRAF6 in Traf6^−/− MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host’s E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.