Bortezomib Rescues Ovariectomy-Induced Bone Loss via SMURF-Mediated Ubiquitination Pathway

A balance between bone formation by osteoblasts and bone resorption by osteoclasts is necessary to maintain bone health and homeostasis. As a cancer of plasma cells, multiple myeloma (MM) is accompanied with rapid bone loss and fragility fracture. Bortezomib has been used as a first-line for treating MM for decades. Recently, the potential protection of bortezomib on osteoporosis (OP) is reported; however, the specific mechanism involving bortezomib-mediated antiosteoporotic effect is undetermined. In the present study, we assessed the effects of in vitro bortezomib treatment on osteogenesis and osteoclastogenesis and the protective effect on bone loss in ovariectomized (OVX) mice. Our results indicated that bortezomib treatment increased osteogenic differentiation of MC3T3-E1 cells as evidenced by increased levels of matrix mineralization and osteoblast-specific markers. In bortezomib-treated bone marrow monocytes (BMMs), osteoclast differentiation was suppressed, substantiated by downregulated tartrate-resistant acid phosphatase- (TRAP-) positive multinucleated cells, areas of actin rings, pit formation, and osteoclast-specific genes. Mechanistically, bortezomib exerted a protective effect against OP through the Smad ubiquitination regulatory factor- (SMURF-) mediated ubiquitination pathway. Furthermore, in vivo intraperitoneal injection of bortezomib attenuated the bone microarchitecture in OVX mice. Accordingly, our findings corroborated that bortezomib might have future applications in the treatment of postmenopausal OP.

Curcumin prevents As3+-induced carcinogenesis through regulation of GSK3β/Nrf2

Background Arsenic (As3+) is a carcinogen with considerable environmental and occupational relevancy. Its mechanism of action and methods of prevention remain to be investigated. Previous studies have demonstrated that ROS is responsible for As3+-induced cell transformation, which is considered as the first stage of As3+ carcinogenesis. The NF-E2 p45-related factor-2 (Nrf2) signaling pathway regulates the cellular antioxidant response, and activation of Nrf2 has recently been shown to limit oxidative damage following exposure to As3+ Methods and results In this study, molecular docking was used to virtually screen natural antioxidant chemical databases and identify molecules that interact with the ligand-binding site of Keap1 (PDB code 4L7B). The cell-based assays and molecular docking findings revealed that curcumin has the best inhibitory activity against Keap1-4L7B. Co-immunoprecipitation (Co-IP) results indicated that curcumin is a potent Keap1 Kelch domain-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. The increased activation of Nrf2 and its target antioxidant genes by curcumin could significantly decrease As3+-generated ROS. Moreover, curcumin induced autophagy in As3+-treated BEAS-2B via inducing autophagy by the formation of a p62/LC-3 complex and increasing autophagic flux by promoting transcription factor EB (TFEB) and lysosome-associated membrane protein 1 (LAMP1) expression. Knockdown of Nrf2 abolished curcumin-induced autophagy and downregulated ROS. Further studies showed that inhibition of autophagosome and lysosome fusion with bafilomycin a1 (BafA1) could block curcumin and prevented As3+-induced cell transformation. These results demonstrated that curcumin prevents As3+-induced cell transformation by inducing autophagy via the activation of the Nrf2 signaling pathway in BEAS-2B cells. However, overexpression of Keap-1 showed a constitutively high level of Nrf2 in As3+-transformed BEAS-2B cells (AsT) is Keap1-independent regulation. Overexpression of Nrf2 in AsT demonstrated that curcumin increased ROS levels and induced cell apoptosis via the downregulation of Nrf2. Further studies showed that curcumin decreased the Nrf2 level in AsT by activating GSK-3β to inhibit the activation of PI3K/AKT. Co-IP assay results showed that curcumin promoted the interaction of Nrf2 with the GSK-3β/β-TrCP axis and ubiquitin. Moreover, the inhibition of GSK-3β reversed Nrf2 expression in curcumin-treated AsT, indicating that the decrease in Nrf2 is due to activation of the GSK-3β/β-TrCP ubiquitination pathway. Furthermore, in vitro and in vivo results showed that curcumin induced cell apoptosis, and had anti-angiogenesis and anti-tumorigenesis effects as a result of activating the GSK-3β/β-TrCP ubiquitination pathway and subsequent decrease in Nrf2. Conclusions Taken together, in the first stage, curcumin activated Nrf2, decreased ROS, and induced autophagy in normal cells to prevent As3+-induced cell transformation. In the second stage, curcumin promoted ROS and apoptosis and inhibited angiogenesis via inhibition of constitutive expression of Nrf2 in AsT to prevent tumorigenesis. Our results suggest that antioxidant natural compounds such as curcumin can be evaluated as potential candidates for complementary therapies in the treatment of As3+-induced carcinogenesis.

Changes in DNA methylation during pregnancy and after delivery

Background Gestational adaptation takes place soon after fertilization and continues throughout pregnancy, while women return to a pre-pregnancy state after delivery and lactation. However, little is known about the role of DNA methylation in the fine tuning of maternal physiology. In this study, we investigated whether and how the DNA methylation pattern changes in the three trimesters and after delivery in ten uncomplicated pregnancies. Results DNA methylation was measured using Human MethylationEPIC BeadChip. There are 14,018 CpG sites with statistically significant changes in DNA methylation over the four time periods (p < .001). Overall, DNA methylation of the non-pregnant state was higher than that of the three trimesters (p < .001), with the protein ubiquitination pathway the top canonical pathway involved. We classified these CpG sites into nine groups according to the changes in the three trimesters. During pregnancy, most CpG sites (61.63%) had subtle or no changes in DNA methylation (Group 9). There were 3,173 (22.64%) CpG sites in Group 7 and 995 (7.1%) CpG sites in Group 8, which were the two groups with DNA methylation changes between the first and second trimesters. The top systems involved in these two groups were associated with embryonic development and organ morphological changes, as shown by the IPA analysis. Conclusion The DNA methylation pattern changes between trimesters, which may be involved in maternal adaptation to pregnancy. Meanwhile, the DNA methylation patterns during pregnancy and in the postpartum period were different, implying that puerperium repair may also act through DNA methylation mechanisms.

WGCNA Identifies Translational and Proteasome-Ubiquitin Dysfunction in Rett Syndrome

Rett Syndrome (RTT) is an X linked neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, resulting in severe cognitive and physical disabilities. Despite an apparent normal prenatal and postnatal development period, symptoms usually present around 6 to 18 months of age. Little is known about the consequences of MeCP2 deficiency at a molecular and cellular level before the onset of symptoms in neural cells, and subtle changes at this highly sensitive developmental stage may begin earlier than symptomatic manifestation. Recent transcriptomic studies of patient induced pluripotent stem cells (iPSC)-differentiated neurons and brain organoids harbouring pathogenic mutations in MECP2, have unravelled new insights into the cellular and molecular changes caused by these mutations. Here we interrogated transcriptomic modifications in RTT patients using publicly available RNA-sequencing datasets of patient iPSCs harbouring pathogenic mutations and healthy control iPSCs by Weighted Gene Correlation Network Analysis (WGCNA). Preservation analysis identified core gene pathways involved in translation, ribosomal function, and ubiquitination perturbed in some MECP2 mutant iPSC lines. Furthermore, differential gene expression of the parental fibroblasts and iPSC-derived neurons revealed alterations in genes in the ubiquitination pathway and neurotransmission in fibroblasts and differentiated neurons respectively. These findings might suggest that global translational dysregulation and proteasome ubiquitin function in Rett syndrome begins in progenitor cells prior to lineage commitment and differentiation into neural cells.

F-box protein MdAMR1L1 regulates ascorbate biosynthesis in apple by modulating GDP-mannose pyrophosphorylase

Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.

Ubiquitin mediated degradation of EGFR by 17 β-estradiol in triple negative MDA-MB-231 (TNBC) breast cancer cells line

Background: Triple Negative Breast Cancer (TNBC) commonly displays Epidermal growth factor receptor (EGFR). Effective EGFR degradation results in suppression of tumor in various models. Studies have addressed the relevance of this strategy in the treatment of TNBC. In the present study, we examined the effect of 17 β-estradiol on EGFR expression in MDA-MB-231 (TNBC) cell line and assessed whether 17 β-estradiol degrades EGFR by ubiquitination pathway. Objectives: To treat MDA-MB-231 cell lines with Cycloheximide with or without 17β-estrdiol to observe whether 17β-estradiol leads to EGFR degradation. To treat with MG-132 to assess whether degradation occurs through ubiquitination pathway. Methods: MDA-MB-231 cells were treated with 17β-estradiol (E2) and EGFR expression was studied by western blotting at different intervals by using Cycloheximide chase. To assess the ubiquitination pathway of degradation of EGFR in MDA-MB-231 cell line, MG-132 was used. Results: EGFR expression was reduced with β-estradiol treatment in MDA-MB-231 cell line with Cycloheximide chase. Upon Treatment with MG-132 and E2, EGFR expression did not reduce, suggesting that Estrogen degrades EGFR by ubiquitination pathway. Conclusion: Estrogen degrades EGFR in MDA-MB-231 cells and this degradation occurs by ubiquitination.

Targeting the MYC Ubiquitination-Proteasome Degradation Pathway for Cancer Therapy

Deregulated MYC overexpression and activation contributes to tumor growth and progression. Given the short half-life and unstable nature of the MYC protein, it is not surprising that the oncoprotein is highly regulated via diverse posttranslational mechanisms. Among them, ubiquitination dynamically controls the levels and activity of MYC during normal cell growth and homeostasis, whereas the disturbance of the ubiquitination/deubiquitination balance enables unwanted MYC stabilization and activation. In addition, MYC is also regulated by SUMOylation which crosstalks with the ubiquitination pathway and controls MYC protein stability and activity. In this mini-review, we will summarize current updates regarding MYC ubiquitination and provide perspectives about these MYC regulators as potential therapeutic targets in cancer.

Culture of Mycobacterium smegmatis in Different Carbon Sources to Induce In Vitro Cholesterol Consumption Leads to Alterations in the Host Cells after Infection: A Macrophage Proteomics Analysis

During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.

USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

Tumor cell dissemination in cancer patients is associated with a significant reduction in their survival and quality of life. The ubiquitination pathway plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions and its dysregulation has been associated with malignant transformation and invasive potential of tumor cells, thus highlighting its value as a potential therapeutic target. In order to identify novel molecular targets of tumor cell migration and invasion we performed a genetic screen with an shRNA library against ubiquitination pathway-related genes. To this end, we set up a protocol to specifically enrich positive migration regulator candidates. We identified the deubiquitinase USP19 and demonstrated that its silencing reduces the migratory and invasive potential of highly invasive breast cancer cell lines. We extended our investigation in vivo and confirmed that mice injected with USP19 depleted cells display increased tumor-free survival, as well as a delay in the onset of the tumor formation and a significant reduction in the appearance of metastatic foci, indicating that tumor cell invasion and dissemination is impaired. In contrast, overexpression of USP19 increased cell invasiveness both in vitro and in vivo, further validating our findings. More importantly, we demonstrated that USP19 catalytic activity is important for the control of tumor cell migration and invasion, and that its molecular mechanism of action involves LRP6, a Wnt co-receptor. Finally, we showed that USP19 overexpression is a surrogate prognostic marker of distant relapse in patients with early breast cancer. Altogether, these findings demonstrate that USP19 might represent a novel therapeutic target in breast cancer.