The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.

Genome-Wide Distribution, Expression and Function Analysis of the U-Box Gene Family in Brassica oleracea L.

The plant U-box (PUB) protein family plays an important role in plant growth and development. The U-box gene family has been well studied in Arabidopsis thaliana, Brassica rapa, rice, etc., but there have been no systematic studies in Brassica oleracea. In this study, we performed genome-wide identification and evolutionary analysis of the U-box protein family of B. oleracea. Firstly, based on the Brassica database (BRAD) and the Bolbase database, 99 Brassicaoleracea PUB genes were identified and divided into seven groups (I-VII). The BoPUB genes are unevenly distributed on the nine chromosomes of B. oleracea, and there are tandem repeat genes, leading to family expansion from the A. thaliana genome to the B. oleracea genome. The protein interaction network, GO annotation, and KEGG pathway enrichment analysis indicated that the biological processes and specific functions of the BoPUB genes may mainly involve abiotic stress. RNA-seq transcriptome data of different pollination times revealed spatiotemporal expression specificity of the BoPUB genes. The differential expression profile was consistent with the results of RT-qPCR analysis. Additionally, a large number of pollen-specific cis-acting elements were found in promoters of differentially expressed genes (DEG), which verified that these significantly differentially expressed genes after self-pollination (SP) were likely to participate in the self-incompatibility (SI) process, including gene encoding ARC1, a well-known downstream protein of SI in B. oleracea. Our study provides valuable information indicating that the BoPUB genes participates not only in the abiotic stress response, but are also involved in pollination.

The proteasome function reporter GFPu accumulates in young brains of the APPswe/PS1dE9 Alzheimer's disease mouse model.

Alzheimer's disease (AD), the most common cause of dementia, is neuropathologically characterized by accumulation of insoluble fibrous inclusions in the brain in the form of intracellular neurofibrillary tangles and extracellular senile plaques. Perturbation of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the pathogenesis of AD. However, studies on UPS functionality with various methods and AD models have achieved non-conclusive results. To get further insight into UPS functionality in AD, we have crossed a well-documented APPswe/PS1dE9 AD mouse model with a UPS functionality reporter, GFPu, mouse expressing green fluorescence protein (GFP) fused to a constitutive degradation signal (CL-1) that facilitates its rapid turnover in conditions of a normal UPS. Our western blot results indicate that GFPu reporter protein was accumulated in the cortex and hippocampus, but not striatum in the APPswe/PS1dE9 AD mouse model at 4 weeks of age, which is confirmed by fluorescence microscopy and elevated levels of p53, an endogenous UPS substrate. In accordance with this, the levels of ubiquitinated proteins were elevated in the AD mouse model. These results suggest that UPS is either impaired or functionally insufficient in specific brain regions in the APPswe/PS1dE9 AD mouse model at a very young age, long before senile plaque formation and the onset of memory loss. These observations may shed new light on the pathogenesis of AD.

let-7 Overexpression leads to an increased fraction of cells in G2/M, direct down-regulation of Cdc34, and stabilization of Wee1 kinase in primary fibroblasts.

microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G(2)/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3'-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3'-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G(2)/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G(2)/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G(2)/M in primary fibroblasts.

The ubiquitin-conjugating enzymes, Ubc4 and Cdc34, mediate cadmium resistance in budding yeast through different mechanisms.

The expression of the genes encoding the ubiquitin-conjugating enzymes, Ubc4, Ubc5, and Ubc7, has been reported to be induced by cadmium in budding yeast. In contrast, we have reported that the overexpression of Cdc34, another ubiquitin-conjugating enzyme, confers resistance to cadmium. In the present study, we examined the effects of overexpression of Ubc4, Ubc5, or Ubc7 on the sensitivity of budding yeast to cadmium. We found that yeast cells that overexpressed Ubc4, but not Ubc5 or Ubc7, showed similar cadmium resistance as yeast cells that overexpressed Cdc34. The ubiquitination levels of cellular proteins were significantly increased by overexpression of Ubc4 as well as by Cdc34. As previously reported, yeast cells overexpressing Cdc34 were resistant to cadmium even in the presence of the proteasome inhibitor MG132. However, the acquired resistance to cadmium by overexpression of Ubc4 was not observed in the presence of MG132. Cdc34 overexpression has been shown to inactivate the transcriptional activity of Met4 by accelerating its ubiquitination and to reduce expression of the MET25 gene, a target gene of Met4. Unlike Cdc34, overexpression of Ubc4 did not affect the expression of the MET25 gene. These findings suggest that the mechanism of acquired resistance to cadmium by overexpression of Ubc4 is different from that of Cdc34 and that Ubc4 confers resistance to cadmium by ubiquitination of proteins other than Met4 and accelerates the degradation of these proteins in the proteasomes.

Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors.

The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.

High-throughput assay to monitor formation of the E2-ubiquitin thioester intermediate.

Targeting components of ubiquitination pathways for drug discovery necessitates the development of high-capacity assays that monitor the ubiquitination process at defined steps of the E1-E2-E3 cascade. This chapter describes the development of an assay based on time-resolved fluorescence to monitor formation of the thioester intermediate between ubiquitin-conjugating enzymes (E2s) and ubiquitin. The methodology is exemplified by an assay tailored for the ubiquitin-conjugating enzyme Cdc34. This assay setup can be easily adapted to other E2s and is suitable to screen small molecule inhibitors of E2-thioester formation in a high-throughput mode.

Expression and assay of HECT domain ligases.

HECT domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin-thioester formation, and substrate ubiquitination.

In vitro reconstitution of SCF substrate ubiquitination with purified proteins.

The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin-dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCF(Cdc4) and its ubiquitin-conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.

Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53.

The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.