Ubiquitin-ligating Enzymes Research Articles

Beyond proteins: Ubiquitylation of lipopolysaccharide to fight bacteria.

Mechanisms and functions of protein ubiquitylation and LPS ubiquitylation. LPS ubiquitylation serves as a scaffold to recruit E3 ligases for the ubiquitylation of the membrane of bacteria. Polyubiquitin coat on the bacterial cell surface is one type of "eat-me" signal recognized by the host cells. The abbreviations used in the figure are Pro, Protein; Ub, Ubiquitin; E1, Ubiquitin-activating enzymes; E2, Ubiquitin-conjugating enzymes; E3, Ubiquitin-ligating enzymes; LPS, lipopolysaccharide; RNF213, Ring Finger Protein 213; and LUBAC, linear ubiquitin chain assembly complex.

Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers.

Protein ubiquitination is involved in many cellular processes, such as protein degradation, DNA repair, and signal transduction pathways. Ubiquitin-conjugating (E2) enzymes of the ubiquitination pathway are associated with various cancers, such as leukemia, lung cancer, and gastric cancer. However, to date, detection of E2 activities is not practicable for capturing the pathological conditions of cancers due to complications related to the enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. Artificial RING fingers (ARFs) were developed to conveniently detect E2 activities during the ubiquitination reaction. ARFs were created by grafting the active sites of ubiquitin-ligating (E3) enzymes onto amino acid sequences with 38 residues. The grafting design downsized E3s to small molecules (ARFs). Such an ARF is a multifunctional molecule that possesses specific E2-binding capabilities and ubiquitinates itself without a substrate. In this review, we discuss the major findings from recent investigations on a new molecular design for ARFs and their simplified detection system for E2 activities. The use of the ARF allowed us to monitor E2 activities using acute promyelocytic leukemia (APL)-derived cells following treatment with the anticancer drug bortezomib. The molecular design of ARFs is extremely simple and convenient, and thus, may be a powerful tool for protein engineering. The ARF methodology may reveal a new screening method of E2s that will contribute to diagnostic techniques for cancers.

Design of aSystem for Monitoring Ubiquitination Activities of E2 Enzymes Using Engineered RING Finger Proteins.

Ubiquitination is a sequential cascade consisting of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. It controls numerous processes such as protein degradation, DNA repair, and signal transduction pathways. E2 enzymes are associated with a variety of diseases such as leukemia, breast cancer, lung cancer, and colorectal cancer. To date, the monitoring of E2 activity for cancer diagnosis is challenging due to its intricate cascade reaction. To surmount this hurdle, we have recently developed a novel strategy for monitoring E2 activities. Here, we describe the concise machinery of ubiquitination with artificial RING finger proteins (ARFs) functioning as E3 enzymes. This machinery enables the simplified monitoring of E2 activities. Furthermore, our system combines a signal accumulation ion-sensitive field-effect transistor biosensor with ARFs, allowing for real-time monitoring of the pathological conditions of cancer cells. The present methodology may lead to novel diagnostic techniques for cancers.

Detection of E2 Activity as a Tumor Marker using an Artificial Ring Finger

Protein ubiquitination is undertaken through an enzymatic cascade comprising ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. The E2 enzymes are associated with various cancers such as leukemia, lung cancer, and breast cancer, and thus, they are attractive targets as tumor markers. In a previous study, we reported the creation of artificial RING fingers that are useful for detecting E2 activity. It was recently reported that a signal accumulation ISFET biosensor (AMIS sensor) allows the detection of proton changes arising during enzyme reactions. In this study, by using the artificial RING fingers and the AMIS sensor, we have succeeded in high-sensitivity quantitative detection of E2 activity. Furthermore, the real-time capturing of E2 activity was successfully achieved using human acute promyelocytic leukemia cells after treatment with the anticancer drug bortezomib. This study allowed the assessment of the pathological conditions of cancer cells. The present method could be applicable to the detection of E2 activity in the blood serum of patients, and therefore, it could be helpful for designing suitable treatment programs for patients. This strategy is extremely simple and convenient, and it opens new possibilities for diagnostic and prognostic methods in tumor development. Patients with cancer could benefit from advances of the quantitative analysis of the E2 activities of the ubiquitination system.

Evolutionary Reshaping of Fungal Mating Pathway Scaffold Proteins

Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating.

A WLM Protein with SUMO-Directed Protease Activity

Covalent modification of eukaryotic proteins by ubiquitin and the distantly related ubiquitin-like protein called SUMO contributes to an extraordinary assortment of biological regulatory mechanisms (2). These attachments have different consequences, depending not only on which of the proteins is attached but also on whether they are in the form of a polymer. Polyubiquitin chains connected by amide (isopeptide) bonds involving certain ubiquitin lysine side chains, for instance, are often signals for directing a substrate to the proteasome for degradation. The ubiquitylation and sumoylation pathways intersect in a variety of ways. An intriguing example is the recently unearthed group of ubiquitin-ligating enzymes called SUMO-targeted ubiquitin ligases (STUbLs), which recognize (poly)SUMO-modified proteins and attach ubiquitin to the conjugates, leading to their degradation by the proteasome (3, 9). Discovery of the STUbLs has raised many interesting questions, such as whether the ubiquitin is attached to the SUMO moiety or the substrate itself in the SUMO-substrate conjugate and how SUMO and ubiquitin are recycled from these conjugates once they have reached the proteasome. In this issue, the study by Mullen et al. (8) throws fresh light on these questions through the characterization of an unusual SUMO-directed protease that may associate with the proteasome and seems able to cleave both ubiquitin and SUMO from proteins.

The SUMO arena goes mitochondrial with MAPL

The SUMO arena goes mitochondrial with MAPL

Mechanistic Insight into Site-Restricted Monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI

A key step in the Fanconi anemia (FA) tumor suppressor pathway is the site-specific monoubiquitination of the FANCD2 protein. Genetic studies indicate that this crucial modification requires eight known FA gene products and the E2-conjugating enzyme Ube2t. Here, we minimally reconstitute this monoubiquitination reaction with Ube2t and the FANCL protein, revealing that monoubiquitination is stimulated by a conserved RWD-like domain in FANCL. Furthermore, addition of the FANCI protein enhances monoubiquitination and also restricts it to the in vivo substrate lysine residue on FANCD2. This work therefore establishes a system that provides mechanistic insight into the functions of FANCL and FANCI in the catalysis of FANCD2 monoubiquitination.

Ubiquitination Controls Life or Death

Tumor necrosis factor (TNF) can trigger cell death or cell survival depending on the context into which the cell receives the signal. O'Donnell et al . show that if lysine 63 ubiquitination of receptor-interacting protein 1 (RIP1) was prevented in Jurkat T cells, the cells exhibited enhanced apoptosis in response to TNF when compared with TNF-treated cells expressing wild-type RIP1. RIP1 ubiquitination was blocked either by expression of a RIP mutant or by expression of dominant-negative forms of the ubiquitin-ligating enzymes TRAF2 (an E3 ubiquitin ligase) or UBC13 (an E2 ubiquitin-conjugating enzyme). The increase in apoptosis did not involve reduced signaling through the nuclear factor κB pathway. Cells expressing wild-type RIP1 exhibited the least apoptosis, whereas cells that were expressing RIP1 and in which TRAF2 activity was inhibited exhibited the most apoptosis, which suggests that the ubiquitination state of RIP1 dictates whether it will generate a proapoptotic signal or a prosurvival signal. RIP1 stimulates apoptosis by activation of caspase 8, and in the RIP1 ubiquitination mutant, RIP1 exhibited increased stimulus-dependent association with procaspase 8. The cells expressing the RIP1 ubiquitination mutant showed increased caspase 8 processing. Thus, ubiquitination of RIP1 appears to prevent its interaction with caspase 8. These results provide an explanation for the conflicting roles assigned to RIP1, as both a proapoptotic and prosurvival mediator. Lysine 63 ubiquitination of RIP1 by TRAF2 switches RIP1 from sending a proapoptotic signal to providing a prosurvival signal. M. A. O'Donnell, D. Legarda-Addison, P. Skountzos, W. C. Yeh, A. T. Ting, Ubiquitination of RIP1 regulates an NF-κB-independent cell-death switch in TNF signaling. Curr. Biol. 17 , 418-424 (2007). [PubMed]

Experimental arthritis inhibits the insulin-like growth factor-I axis and induces muscle wasting through cyclooxygenase-2 activation

Chronic arthritis induces cachexia associated with an inhibition of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system and an activation of the E3 ubiquitin-ligating enzymes muscle atrophy F-box (MAFbx) and muscle Ring finger 1 (MuRF1) in the skeletal muscle. The aim of this work was to study the role of cyclooxygenase (COX)-2 in chronic arthritis-induced cachexia. Arthritis was induced in rats by Freund's adjuvant injection, and the effects of two COX inhibitors (indomethacin, a nonspecific inhibitor, and meloxicam, a selective COX-2 inhibitor on pituitary GH and on liver and serum IGF-I levels) were tested. Arthritis decreased body weight gain and GH and liver IGF-I gene expression. In the arthritic rats, both inhibitors, indomethacin and meloxicam, prevented the inhibitory effect of arthritis on body weight gain. Indomethacin and meloxicam administration to arthritic rats increased pituitary GH and liver IGF-I mRNA as well as serum levels of IGF-I. These data suggest that induction of COX-2 during chronic inflammation is involved in the inhibition of the GH-IGF-I axis and in the body weight loss. In the gastrocnemius muscle, arthritis increased the gene expression of tumor necrosis factor (TNF)-alpha, the E3 ubiquitin-ligating enzymes MAFbx and MuRF1, as well as of IGF-I and IGF-binding protein-5 (IGFBP-5). Inhibition of COX-2 by meloxicam administration increased gastrocnemius weight and decreased MAFbx, MuRF1, TNF-alpha, and IGFBP-5 gene expression. In summary, our data indicate that chronic arthritis-induced cachexia and muscle wasting are mediated by the COX-2 pathway resulting in a decreased GH-IGF-I secretion and increased expression of MAFbx and MuRF1 mRNA.

Tumour necrosis factor blockade did not prevent the increase of muscular muscle RING finger-1 and muscle atrophy F-box in arthritic rats

Chronic inflammation is associated with a decrease in body weight and cachexia, which is characterized by anorexia and skeletal muscle wasting. The expression of atrogens muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) are increased in muscle atrophy and it is known that tumour necrosis factor (TNF) regulates skeletal muscle loss through TNF receptor p55 (TNFRI). The aim of this study was to examine the effect of polyethylene glycol linked to soluble TNFRI (PEG-sTNFRI) on gene expression of the atrogens MuRF-1 and MAFbx in skeletal muscle of arthritic rats. Rats were injected with Freund's adjuvant and, 15 days later, arthritic and control rats were injected daily with PEG-sTNFRI (1 mg/kg, s.c.) or saline for 8 days. Arthritis decreased body weight gain, the weight of skeletal muscle and adipose mass. PEG-sTNFRI administration increased body weight gain and adipose mass of arthritic rats; however, it did not modify the skeletal muscle weight. The gene expression of TNF-alpha, MuRF1 and MAFbx, IGF-I and IGFBP-5 were increased in the skeletal muscle of arthritic rats, and the administration of PEG-sTNFRI did not modify these parameters. These data suggest that the anti-TNF agent PEG-sTNFRI did not prevent the increase in E3 ubiquitin-ligating enzymes, MuRF1 and MAFbx, gene expression in the skeletal muscle of arthritic rats.

Cell-free ubiquitination of cell cycle regulators in budding yeast extracts

Publisher Summary This chapter discusses the cell-free ubiquitination of cell cycle regulators in budding yeast extracts. Proteolysis of key cell cycle-regulatory molecules is one method of ensuring the irreversibility of phase transitions within the eukaryotic cell cycle. Degradation of such molecules is typically mediated by the ubiquitin pathway, and requires the concerted action of a complex array of proteins including ubiquitin-activating (El), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. The action of these enzymes results in the assembly of multiubiquitin chains on the substrate, which is then targeted to the 26S proteosome where it is cleaved into small peptides. To define the mechanism of action of the ubiquitin-dependent proteolyric pathways that regulate the eukaryotic cell cycle, one needs to have cell-free systems that faithfully reproduce these reactions.

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (n > approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.