Mind Bomb Research Articles

One RING to rule them all: Mind bomb-1 ccRING3 controls Notch signaling

In this issue of Structure, Cao etal.1 use X-ray crystallography, biochemical, and genetic studies to define the key role of the Mind bomb-1 ccRING3 domain in triggering Notch signaling, and they demonstrate that ccRING3-mediated dimerization is a key step in ligand activation.

Structural requirements for activity of Mind bomb1 in Notch signaling

Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

USP18 Antagonizes Pyroptosis by Facilitating Selective Autophagic Degradation of Gasdermin D.

As a key executioner of pyroptosis, Gasdermin D (GSDMD) plays a crucial role in host defense and emerges as an essential therapeutic target in the treatment of inflammatory diseases. So far, the understanding of the mechanisms that regulate the protein level of GSDMD to prevent detrimental effects and maintain homeostasis is currently limited. Here, we unveil that ubiquitin-specific peptidase 18 (USP18) works as a negative regulator of pyroptosis by targeting GSDMD for degradation and preventing excessive innate immune responses. Mechanically, USP18 recruits E3 ubiquitin ligase mind bomb homolog 2 (MIB2) to catalyze ubiquitination on GSDMD at lysine (K) 168, which acts as a recognition signal for the selective autophagic degradation of GSDMD. We further confirm the alleviating effect of USP18 on LPS-triggered inflammation invivo. Collectively, our study demonstrates the role of USP18 in regulating GSDMD-mediated pyroptosis and reveals a previously unknown mechanism by which GSDMD protein level is rigorously controlled by selective autophagy.

Gutless Helper-Dependent and First-Generation HAdV5 Vectors Have Similar Mechanical Properties and Common Transduction Mechanisms.

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

N6-methyladenosine-modified MIB1 promotes stemness properties and peritoneal metastasis of gastric cancer cells by ubiquitinating DDX3X.

Peritoneal metastasis (PM), one of the most typical forms of metastasis in advanced gastric cancer (GC), indicates a poor prognosis. Exploring the potential molecular mechanism of PM is urgently necessary, as it has not been well studied. E3 ubiquitin ligase has been widely established to exert a biological function in various cancers, but its mechanism of action in GC with PM remains unknown. The effect of MIB1 on PM of GC was confirmed in vitro and in vivo. Co-immunoprecipitation (Co-IP) and mass spectrometry demonstrated the association between MIB1 and DDX3X. Western blot, flow cytometry and immunofluorescence determined that DDX3X was ubiquitylated by MIB1 and promoted stemness. We further confirmed that METTL3 promoted the up-regulation of MIB1 by RNA immunoprecipitation (RIP), luciferase reporter assay and other experiments. We observed that the E3 ubiquitin ligase Mind bomb 1 (MIB1) was highly expressed in PMs, and patients with PM with high MIB1 expression showed a worse prognosis than those with low MIB1 expression. Mechanistically, our study demonstrated that the E3 ubiquitin ligase MIB1 promoted epithelial-mesenchymal transition (EMT) progression and stemness in GC cells by degrading DDX3X. In addition, METTL3 mediated m6A modification to stabilize MIB1, which required the m6A reader IGF2BP2. Our study elucidated the specific molecular mechanism by which MIB1 promotes PM of GC, and suggested that targeting the METTL3-MIB1-DDX3X axis may be a promising therapeutic strategy for GC with PM.

Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death.

Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.

Sanggenon C inhibits cell proliferation and induces apoptosis by regulating the MIB1/DAPK1 axis in glioblastoma.

Sanggenon C (SC), a herbal flavonoid extracted from Cortex Mori, has been mentioned to possess more than one treasured organic properties. However, the molecular mechanism of its anti-tumor impact in glioblastoma (GBM) remains unclear. In this study, we reported that SC displayed a GBM-suppressing impact in vitro and in vivo with no apparent organ toxicity. SC dramatically suppressed cell proliferation-induced cell apoptosis in GBM cells. Mechanistically, we unveiled that SC modulated the protein expression of death associated protain kinase 1 (DAPK1) by controlling the ubiquitination and degradation of DAPK1. Quantitative proteomic and Western blot analyses showed that SC improved DAPK1 protein degradation via decreasing the expression of E3 ubiquitin ligase Mindbomb 1 (MIB1). More importantly, the effects of SC on cell proliferation and apoptosis of GBM cells have been in part reversed through DAPK1 downregulation or MIB1 overexpression, respectively. These results indicated that SC might suppress cell proliferation and induce cell apoptosis by decreasing MIB1-mediated DAPK1 degradation. Furthermore, we found that SC acted synergistically with temozolomide (TMZ), an anti-cancer drug used in GBM, resulting in elevated chemotherapeutic sensitivity of GBM to TMZ. Collectively, our data suggest that SC might be a promising anti-cancer agent for GBM therapy.

The E3 ubiquitin ligase MIB1 suppresses breast cancer cell migration through regulating CTNND1 protein level

Breast cancer is one of the most common invasive cancers among women. The leading cause of difficulty in treating breast cancer patients is metastasis. Because cell migration is closely related to breast cancer metastasis, elucidating the detailed mechanism by which breast cancer cells promote their migration is crucial for improving the prognosis of patients. In this study, we investigated the relationship between breast cancer cell migration and Mind bomb1 (MIB1), an E3 ubiquitin ligase. We found that the downregulation of MIB1 promotes the cell migration of MCF7, a breast cancer-derived cell line. Furthermore, knockdown of MIB1 caused a reduction in CTNND1 and thereby impaired E-cadherin membrane localization in the cell boundary region. Taken together, our data suggest that MIB1 might play a role in suppressing breast cancer cell migration.

Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2

Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identify the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulates the effects of FAT1 depletion and causes decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis.

PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.

Programmed death-ligand 1 (PD-L1), a critical immune checkpoint ligand, is a transmembrane protein synthesized in the endoplasmic reticulum of tumor cells and transported to the plasma membrane to interact with programmed death 1 (PD-1) expressed on T cell surface. This interaction delivers coinhibitory signals to T cells, thereby suppressing their function and allowing evasion of antitumor immunity. Most companion or complementary diagnostic devices for assessing PD-L1 expression levels in tumor cells used in the clinic or in clinical trials require membranous staining. However, the mechanism driving PD-L1 translocation to the plasma membrane after de novo synthesis is poorly understood. Herein, we showed that mind bomb homolog 2 (MIB2) is required for PD-L1 transportation from the trans-Golgi network (TGN) to the plasma membrane of cancer cells. MIB2 deficiency led to fewer PD-L1 proteins on the tumor cell surface and promoted antitumor immunity in mice. Mechanistically, MIB2 catalyzed nonproteolytic K63-linked ubiquitination of PD-L1, facilitating PD-L1 trafficking through Ras-associated binding 8-mediated (RAB8-mediated) exocytosis from the TGN to the plasma membrane, where it bound PD-1 extrinsically to prevent tumor cell killing by T cells. Our findings demonstrate that nonproteolytic ubiquitination of PD-L1 by MIB2 is required for its transportation to the plasma membrane and tumor cell immune evasion.

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Acid ceramidase targeting pyruvate kinase affected trypsinogen activation in acute pancreatitis

BackgroundAcute pancreatitis is the sudden inflammation of the pancreas. Severe cases of acute pancreatitis are potentially fatal and have no specific treatment available. Premature trypsinogen activation could initiate acute pancreatitis. However, the mechanism underlying premature trypsinogen activation is not fully understood.MethodsIn this research, a primary pancreatic acinar cell or mouse acute pancreatitis model was constructed. The effect of acid ceramidase (ASAH1), which is responsible for sphingosine production, was investigated in trypsinogen activation in vitro and in vivo. Meanwhile, the proteins regulating ASAH1 or binding to sphingosine were also detected by co-immunoprecipitation followed by mass spectrometry.ResultsThe results showed that ASAH1 increased in acute pancreatitis. Increased ASAH1 promoted the activation of trypsinogen and cathepsin B. On the contrary, ASAH1 downregulation inhibited trypsinogen and cathepsin B. Meanwhile, ASAH1 regulated the activity of trypsin and cathepsin B through sphingosine. Additionally, E3 ligase Mind bomb homolog 1 (MIB1) decreased in acute pancreatitis resulting in the decreased binding between MIB1 and ASAH1. Exogenous MIB1 diminished the elevation in trypsin activity induced by acute pancreatitis inducer. ASAH1 increased owing to the inhibition of the proteasome degradation by MIB1. In acute pancreatitis, sphingosine was found to bind to pyruvate kinase. Pyruvate kinase activation could reduce trypsinogen activation and mitochondrial reactive oxygen species (ROS) production induced by sphingosine.ConclusionsIn conclusion, during the process of acute pancreatitis, MIB1 downregulation led to ASAH1 upregulation, resulting in pyruvate kinase inhibition, followed by trypsinogen activation.

Mind bomb 2 promotes cell migration and epithelial structure by regulating adhesion complexes and the actin cytoskeleton

Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, we found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, we identified components of Mib2 complexes, which include E-cadherin and α- and β-catenins, as well as actin regulators. We also found that three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. We conclude that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

E3 ubiquitin-protein ligase 2 inhibits cell proliferation, migration, and invasion of non-small cell lung cancer through ubiquitination of Notch1

This study aimed to explore the role of MIB2 in non-small cell lung cancer (NSCLC) and the underlying mechanism. Quantitative real-time PCR (QRT-PCR) and western blot were first performed to detect MIB2 expression in tumor tissues obtained from NSCLC patients (n = 30) and NSCLC cells, respectively. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and transwell assays were then used to examine the effect of MIB2 on the proliferation, migration and invasion of NSCLC cells. Western blot was further performed to examine the effect of Mind bomb 2 (MIB2), an E3 ligase on Notch1 protein and its ubiquitination. MIB2 was significantly down-regulated in NSCLC tissues and cells, both in mRNA and protein level. MIB2 also note worthily inhibited the proliferation, migration, and invasion of NSCLC cells. Furthermore, MIB2 only down-regulated Notch1 protein level, while facilitated the ubiquitination of Notch1. Additionally, Notch1 significantly relieved the repressed proliferation, migration and invasion of NSCLC cells induced by MIB2. Conclusively, MIB2 inhibited cell proliferation, migration and invasion via inducing Notch1 ubiquitination and degradation in NSCLC.

E3 ubiquitin ligase mind bomb 1 overexpression reduces apoptosis and inflammation of cardiac microvascular endothelial cells in coronary microvascular dysfunction

BackgroundThe apoptosis and inflammation in cardiac microvascular endothelial cells (CMECs) promote the development of coronary microvascular dysfunction (CMD). The present study aimed to explore the role of E3 ubiquitin ligase mind bomb 1 (MIB1) in the apoptosis and inflammation in CMECs during CMD. MethodsIn vivo, CMD in rats was induced by sodium laurate injection. In vitro, rat primary CMECs were stimulated by homocysteine (Hcy). The apoptosis of CMECs was measured using flow cytometry. The inflammation of CMECs was evaluated by the level of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). The interplay between MIB1 and mitogen-activated protein kinase kinase kinase 5 (map3k5, also called ASK1) was measured using Co-immunoprecipitation. ResultsMIB1 expression was decreased and ASK1 expression was increased in the heart tissues of CMD rats and Hcy-treated CMECs. MIB1 overexpression decreased fibrinogen-like protein 2 (FGL2) secretion, inflammation, and apoptosis induced by Hcy in CMECs. Meanwhile, MIB1 overexpression decreased the protein levels of ASK1 and p38, while not affected ASK1 mRNA levels. The following mechanism experiments revealed that MIB1 downregulated ASK1 expression by increasing its ubiquitination. ASK1 overexpression reversed the inhibitory effect of MIB1 on FGL2 secretion, apoptosis, inflammation, and p38 activation in Hcy-treated CMECs. In CMD rats, MIB1 overexpression partly retarded CMD progression, manifesting as increased coronary capillary density and decreased microthrombi formation. ConclusionMIB1 overexpression relieved apoptosis and inflammation of CMECs during CMD by targeting the ASK1/p38 pathway.

The Australasian Cell Death Society (ACDS): celebrating 50 years of Australasian cell death research

The Australasian Cell Death Society (ACDS): celebrating 50 years of Australasian cell death research

MicroRNA‑195‑5p is associated with cell proliferation, migration and invasion in prostate cancer and targets MIB1.

Mind bomb 1 (MIB1) is a well-known E3 ubiquitin ligase. MicroRNAs (miRNAs/miRs) have been found to serve important functions in cancer cell physiology. However, the clinical significance and biological function of MIB1 and miRNAs in prostate cancer (PCa) are yet to be fully elucidated. The current study predicted the interaction between MIB1 and miR-195-5p using TargetScan, and the results were confirmed by performing a dual-luciferase reporter assay. The mRNA expression level of MIB1 and miR-195-5p in PCa and adjacent normal tissues, and PCa cell lines was detected using reverse transcription-quantitative PCR. Cell Counting Kit-8 and Transwell assays were used to measure the proliferation, and migration and invasion of VCaP and DU145 PCa cell lines, respectively, while western blot analysis was used to detect the protein expression level of MIB1. The results revealed that the mRNA expression level of MIB1 was increased, while the mRNA expression level of miR-195-5p was decreased in PCa tissues (P<0.001 and P<0.01, respectively) and in various cell lines, including PC-3 (P<0.001 and P<0.05, respectively), VCaP (P<0.001 and P<0.01, respectively), 22Rv1 (P<0.001 and P<0.05, respectively), DU145 (P<0.001 and P<0.01, respectively) and LNCaP (P<0.001 and P<0.05, respectively). miR-195-5p mimics rescued the inhibitory effects caused by knockdown of MIB1 on cell proliferation, migration and invasion in the VCaP and DU145 cell lines. In addition, MIB1 overexpression restored the miR-195-5p overexpression-induced repression of cell proliferation and invasion. The current study revealed that the MIB1 gene was an effector of cell proliferation, migration and invasion in PCa cell lines. Furthermore, miR-195-5p may regulate PCa cell proliferation and invasion by regulating MIB1, indicating its potential therapeutic application for PCa in the future.

Participation of Mind Bomb-2 in Sevoflurane Anesthesia Induces Cognitive Impairment in Aged Mice via Modulating Ferroptosis.

Postoperative cognitive dysfunction (POCD) is a complication of the central nervous system (CNS) often occurred after surgery or anesthesia in the elder patients. Mind bomb-2 (MIB2) has been reported to modulate neuronal functions. Here, we aimed to study whether MIB2 exerts roles in the effects of sevoflurane anesthesia on mice hippocampal neurons and function, and how. Aging male C57BL/6 mice were subjected to sevoflurane administration, and primary hippocampal neurons were adopted to study sevoflurane effects in vitro. Western blotting and immunohistochemistry assay were used to study the protein expression of MIB2. CCK-8 assay and propidium iodide (PI) staining were performed to evaluate cell viability and cell death, respectively. Ferroptosis-related indicators malondialdehyde (MDA), glutathione (GSH), and iron levels were checked through indicated ELISA kits. Co-immunoprecipitation was adopted to study the binding effects of MIB2 to GPX4. We found that sevoflurane anesthesia increased MIB2 expression in mice hippocampus tissues and neurons. Knockdown of MIB2 alleviated neuron death and ferroptosis induced by sevoflurane exposure. Downregulated MIB2 enhanced GPX4 stability and reduced its ubiquitination. MIB2 was verified to bind to GPX4. The effects of MIB2 knockdown on the neuron death and ferroptosis can be reversed by further siGPX4 transfection. In vivo results also showed that MIB2 knockdown reduced hippocampal neuron death, ferroptosis, and cognitive impairments in the sevoflurane-exposed mice. Taking all together, downregulation of MIB2 could alleviate the sevoflurane-anesthesia-induced cognitive dysfunction and neuron injury through reducing ferroptosis via GPX4. Our results also provide novel directions for POCD treatment using anti-MIB2-related drugs or strategies.