The p105 NF-ĸB precursor is a pseudo substrate of the ubiquitin ligase FBXO7, and its binding to the ligase stabilizes it and results in stimulated cell proliferation.

The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.

Identification of UBact, a ubiquitin-like protein, along with other homologous components of a conjugation system and the proteasome in different gram-negative bacteria.

Systems analogous to the eukaryotic ubiquitin-proteasome system have been previously identified in Archaea, and Actinobacteria (gram-positive), but not in gram-negative bacteria. Here, we report the bioinformatic identification of a novel prokaryotic ubiquitin-like protein, which we name UBact. The phyletic distribution of UBact covers at least five gram-negative bacterial phyla, including Nitrospirae, Armatimonadetes, Verrucomicroba, Nitrospinae, and Planctomycetes. Additionally, it was identified in seven candidate (uncultured) phyla and one Archaeon. UBact might have been overlooked because only few species in the phyla where it is found have been sequenced. In most of the species where we identified UBact, its neighbors in the genome code for proteins homologous to those involved in conjugation and/or degradation of Pup and Pup-tagged substrates. Among them are PafA-, Dop-, Mpa- and proteasome-homologous proteins. This gene association as well as UBact's size and conserved C-terminal G[E/Q] motif, strongly suggest that UBact is used as a conjugatable tag for degradation. With regard to its C-terminus, UBact differs from ubiquitin and most ubiquitin-like proteins (including the mycobacterial Pup) in that it lacks the characteristic C-terminal di-glycine motif, and it usually ends with the sequence R[T/S]G[E/Q]. The phyla that contain UBact are thought to have diverged over 3000 million years ago, indicating that either this ubiquitin-like conjugation system evolved early in evolution or that its occurrence in distant gram-negative phyla is due to multiple instances of horizontal gene transfer.

On the linkage between the ubiquitin-proteasome system and the mitochondria.

Several metabolic pathways critical for cellular homeostasis occur in the mitochondria. Because of the evolution of mitochondria and their physical separation, these pathways have traditionally been thought to be free from regulation by the ubiquitin-proteasome system. This perception has recently been challenged by evidence for the presence of ubiquitin system components in the mitochondria. Furthermore, it has been shown that certain mitochondrial proteins are conjugated by ubiquitin, and some of them are degraded by the proteasome. Of particular interest is the finding that some of these proteins are localized to the inner membrane and matrix, which rules out that their targeting is mediated by the cytosolic ubiquitin system. However, the extent of the involvement of the ubiquitin system in mitochondrial regulation is not known. The present study addresses this surprising finding, employing several independent approaches. First, we identified reported ubiquitin conjugates in human and yeast mitochondria and found that a large fraction of the mitochondrial proteome (62% in human) is ubiquitinated, with most proteins localized to the inner membrane and matrix. Next, we searched the literature and found that numerous ubiquitin system components localize to the mitochondria and/or contain mitochondrial targeting sequences. Finally, we identified reported protein-protein interactions between ubiquitin system components and mitochondrial proteins. These unexpected findings suggest that mitochondrial regulation by the ubiquitin system is fundamental and may have broad biomedical implications.

Ubiquitination of specific mitochondrial matrix proteins.

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins.

Catechol metabolites of endogenous estrogens induce redox cycling and generate reactive oxygen species in breast epithelial cells.

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1).

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC(50) = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2(-/-) mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity.

Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase.

Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2µM, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5µM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)≈6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.