Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme.
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ABSTRACT:
SUBMITTER: Nouspikel T
PROVIDER: S-EPMC1621053 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During sepsis, systemic inflammation is observed and is associated with multiple organ failure. Activation of NF-κB is crucial for inducing inflammation, which is controlled by degradation of inhibitor molecules (IκB). The ubiquitination proteasome pathway is responsible for the regulation of protein turnover. In this study, we hypothesized that administration of 4[4-(5-nitro-furan-2-ylmethylene)-3, -dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41), an inhibitor of ubiquitination, could reduce inflammation and organ injury in septic mice. PYR-41 prevented the reduction of IκB protein levels and inhibited release of tumor necrosis factor (TNF)-α in mouse macrophage RAW264.7 cells at 4 h after lipopolysaccharide stimulation dose-dependently. Male C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. PYR-41 (5 mg/kg) or dimethyl sulfoxide in saline (vehicle) was injected intravenously immediately after CLP. At 20 h after CLP, PYR-41 treatment significantly decreased serum levels of proinflammatory cytokines (TNF-α, interleukin [IL]-1β, and IL-6) and organ injury markers (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). PYR-41 significantly improved microscopic structure, and reduced myeloperoxidase activity, number of apoptotic cells and caspase-3 degradation in the lungs of septic mice. The reduced protein levels of IκB in the lungs after CLP were restored by PYR-41 treatment. PYR-41 inhibited the expression of cytokines (IL-1β and IL-6), chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2), and inflammatory mediators (cyclooxygenase-2 and inducible nitric oxide synthase) in the lungs of septic mice. Importantly, PYR-41 significantly increased 10-day survival in septic mice from 42% to 83%. Therefore, targeting ubiquitination by PYR-41 to inhibit NF-κB activation may represent a potential strategy of sepsis therapeutics.
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Project description:Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
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Project description:E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys(250)) is part of the adenylation domain in an alpha-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.
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Project description:The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.
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Project description:Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and ?-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.
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