Impact of Ubiquitination Signaling Pathway Modifications on Oral Carcinoma

Among intra-cellular homeostasis mechanisms, ubiquitination plays a critical role in protein metabolism regulation by degrading proteins via activating a broad spectrum of ubiquitin chains. In fact, ubiquitination and sumoylation signaling pathways are characterized by increased complexity regarding the molecules and their interactions. The Ubiquitin-Proteasome System (Ub-PS) recognizes and targets a broad spectrum of protein substrates. Ubiquitin conjugation modifies each substrate protein determining its biochemical fate (degradation). A major functional activity of Ub-PS is autophagy mechanism regulation. Interestingly, Ub-PS promotesall stages of bulk autophagy (initiation, execution, and termination). Autophagy is a crucial catabolic process that provides protein degradation and for this reason the interaction with Ub-PS is crucial. Furthermore, ubiquitination controls and regulates specific types of protein targets. Ub-PS is also involved in oxidative cellular stress and DNA damage response. Additionally, the functional role of Ub-PS in ribosome machinery regulation seems to be crucial. Concerning carcinogenesis, Ub-PS is involved in malignant disease development and progression by negatively affecting the corresponding TGF-B-, MEEK/MAPK/ERK-JNK- dependent signaling pathways. In the current review article, we describe the role of Ub-PSbiochemicalmodifications and alterations in oral squamous cell carcinoma (OSCC).

Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO–NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1−/− mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.

RING domains act as both substrate and enzyme in a catalytic arrangement to drive self-anchored ubiquitination

Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation.

Synthesis and delivery of a stable phosphorylated ubiquitin probe to study ubiquitin conjugation in mitophagy

Synthesis, delivery and cellular conjugation analysis of differentially phosphorylated ubiquitin probes by parkin E3 ligase during mitophagy.

Cleaning the molecular machinery of cells via proteostasis, proteolysis and endocytosis selectively, effectively, and precisely: intracellular self-defense and cellular perturbations

Schematic of the regulation of the ubiquitin-protein ligases and ubiquitylation, a dynamic cellular process for stability, and induced protein folding; the ubiquitin-conjugation machinery for accurate surveillance, cell cycle arrest, DNA damage and repair, senescence, and apoptosis.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

The Ubiquitin Proteasome System in Periodontal Disease: A Comprehensive Review

The turnover of intracellular proteins is a highly selective and regulated process. This process is responsible for avoiding injury and irreparable breakdown of cellular constituents. Its impairment disrupts cellular stability, integrity, and homeostasis. The ubiquitin-proteasome system (UPS) is responsible for this programmed degradation of most intracellular proteins. This process involves a cascade of enzymes that involves the ubiquitin conjugation to a target substrate protein, its recognition and degradation by the proteasome. The turn-over of intracellular proteins is a non-stop ubiquitous process that regulates a series of mechanisms, for instance transcription, translation, endocytosis. In addition, proteasome act by releasing peptides that may serve to other purposes, such as antigen presentation in immune actions and enzymatic flagging toward biosynthesis and gluconeogenesis. The role of the UPS impairment in periodontal diseases is gaining growing. This acquaintance might contribute to the development of novel therapeutic applications. Thus, this review focuses on the latest progresses on the role of the UPS and its signaling pathways in Periodontal Medicine. Furthermore, we discuss the potential of UPS-based drugs development to be used in periodontal disease therapy.

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.