T cells efficiently respond to foreign antigens to mediate immune responses against infections but are tolerant to self-tissues. Defect in T cell activation is associated with severe immune deficiencies, whereas aberrant T cell activation contributes to the pathogenesis of diverse autoimmune and inflammatory diseases. An emerging mechanism that regulates T cell activation and tolerance is ubiquitination, a reversible process of protein modification that is counter-regulated by ubiquitinating enzymes and deubiquitinases (DUBs). DUBs are isopeptidases that cleave polyubiquitin chains and remove ubiquitin from target proteins, thereby controlling the magnitude and duration of ubiquitin signaling. It is now well recognized that DUBs are crucial regulators of T cell responses and serve as potential therapeutic targets for manipulating immune responses in the treatment of immunological disorders and cancer. This review will discuss the recent progresses regarding the functions of DUBs in T cells.
Cell Differentiation ; drug effects ; Deubiquitinating Enzymes ; metabolism ; Drug Discovery ; Humans ; Neoplasms ; drug therapy ; pathology ; Signal Transduction ; T-Lymphocytes ; physiology ; Ubiquitination ; drug effects ; physiology

Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Drug Design ; Drug Discovery ; Enzyme Inhibitors ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; enzymology ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

This study examined the mechanism of the inhibitory effect of parthenolide (PTL) on the activity of NF-κB in multiple myeloma (MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6 (TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.
Apoptosis ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; NF-kappa B ; antagonists & inhibitors ; blood ; Sesquiterpenes ; pharmacology ; TNF Receptor-Associated Factor 6 ; metabolism ; Transcription Factor RelA ; metabolism ; Ubiquitination ; drug effects

During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-kappaB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-kappaB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-kappaB activation in TNFalpha-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFalpha, and the subsequent expression of NF-kappaB-dependent genes and the levels of NF-kappaB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFalpha-induced expression of an NF-kappaB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1beta). The degradation of inhibitor of NF-kappaB alpha (IkappaBalpha) and the nuclear translocation of NF-kappaB were also delayed by H2O2 treatment, whereas IkappaBalpha phosphorylation and IkappaB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IkappaBalpha poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-kappaB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IkappaBalpha in HEK293 cells.
Active Transport, Cell Nucleus ; Cell Nucleus/metabolism ; Enzyme Activation/drug effects ; HEK293 Cells ; Humans ; Hydrogen Peroxide/*pharmacology ; I-kappa B Kinase/antagonists & inhibitors/*metabolism ; Phosphorylation/drug effects ; Protein Transport ; Tumor Necrosis Factor-alpha/*pharmacology ; Ubiquitination/*drug effects

OBJECTIVETo probe the effects of dexamethasone (DEX) combined with histone deacetylase (HDAC) inhibitor vorinostat on inhibiting proliferation and inducing differentiation and apoptosis in Kasumi-1 leukemia cells, and its possible mechanisms in order to provide a theoretical basis for the treatment of AML1-ETO positive AML.

METHODSThe cell survival, differentiation and apoptosis rates were tested by MTT or flow cytometry analysis after Kasumi-1 cells were treated by DMSO, DEX (20 nmol/L), vorinostat (1 μmol/L) or DEX (20 nmol/L) in combination with vorinostat (1 μmol/L). WB and IP-WB were performed to detect AML1-ETO and its ubiquitination.

RESULTSTreatment with the combination of DEX and vorinostat for 48 h led to statistically significant differences of inhibited proliferation [(42.06±8.20)%], increased differentiation [(52.83±8.97)%] and apoptosis [(52.92±2.53)%] of Kasumi-1 cells when compared with vorinostat [(33.82±9.41)%, (43.93±9.04)% and (42.98±3.01)%, respectively], DEX [(17.30±3.49)%, (22.53±4.51)% and (19.57±2.17)%, respectively] or control [(6.96±0.39)%, (21.73±2.03)% and (6.96±0.39)%, respectively]. Also significant ubiquitination and decreased AML1-ETO protein in Kasumi-1 cells after the combination treatment over single agent or control were observed.

CONCLUSIONThe results indicated that DEX and vorinostat could synergistically inhibit the Kasumi-1 cells proliferation, induce Kasumi-1 cells differentiation and apoptosis through ubiquitination and degradation of AML1-ETO.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Core Binding Factor Alpha 2 Subunit ; metabolism ; Dexamethasone ; pharmacology ; Drug Synergism ; Humans ; Hydroxamic Acids ; pharmacology ; Oncogene Proteins, Fusion ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Ubiquitination

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARΓ. NEDD4-1 overexpression leads to PPARγ ubiquitination and reduced expression of PPARγ. Conversely, knockdown of NEDD4-1 by specific siRNAs abolishes PPARΓ ubiquitination. These data indicate that NEDD4-1 is the E3 ubiquitin ligase responsible for PPARγ ubiquitination. Here, we show that NEDD4-1 delays cellular senescence by degrading PPARΓ expression. Taken together, our data show that E2 could upregulate SIRT1 expression via promoting the PPARΓ ubiquitination-proteasome degradation pathway to delay the process of cell senescence.
Amino Acid Motifs ; Animals ; Cellular Senescence ; Down-Regulation ; drug effects ; Endosomal Sorting Complexes Required for Transport ; antagonists & inhibitors ; genetics ; metabolism ; Estradiol ; pharmacology ; Female ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nedd4 Ubiquitin Protein Ligases ; PPAR gamma ; genetics ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; Protein Structure, Tertiary ; RNA Interference ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Ubiquitin ; metabolism ; Ubiquitin-Protein Ligases ; antagonists & inhibitors ; genetics ; metabolism ; Ubiquitination ; drug effects ; Up-Regulation ; drug effects

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
DNA Damage ; drug effects ; DNA Repair ; genetics ; DNA Replication ; genetics ; DNA-Binding Proteins ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Endopeptidases ; genetics ; Gene Knockout Techniques ; Humans ; Hydrogen Peroxide ; toxicity ; NEDD8 Protein ; genetics ; Oxidative Stress ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Ubiquitination ; genetics ; Ultraviolet Rays