Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

miR-4454 Promotes Hepatic Carcinoma Progression by Targeting Vps4A and Rab27A.

Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-ß1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-ß1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1.

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.

Inhibiting USP8 overcomes hepatocellular carcinoma resistance via suppressing receptor tyrosine kinases.

The ubiquitin-specific protease 8 (USP8) is a prototypic multidomain deubiquitinating enzyme with pleiotropic functions. We investigated the role of USP8 in hepatocellular carcinoma (HCC) by analyzing expression patterns of USP8 in HCC patients, and evaluating its functions and underlying signaling. Among 20 HCC patients investigated, we found that USP8 protein upregulation was a common phenomenon (17 out of 20) in HCC compared to normal liver tissue. Furthermore, the upregulation of USP8 was not associated with any clinicopathology. USP8 inhibition via genetic and pharmacological approaches resulted in growth inhibition and apoptosis induction in both sensitive and doxorubicin-resistant HCC cells. Of note, USP8 inhibition significantly enhanced doxorubicin or sorafenib's efficacy in HCC cells and mouse models. We further found that USP8 inhibition decreased levels of multiple receptor tyrosine kinases (RTKs) by ~90%, such as epidermal growth factor receptor (EGFR) and c-Met. Consistently, the downstream signaling regulated by RTKs was disrupted in HCC cells after USP8 inhibition, as shown by the decreased p-Akt, p-STAT3 and p-Raf. Our findings demonstrate that USP8 is a novel therapeutic target in HCC. Inhibiting USP8 has potential to overcome current drug resistance, particularly on HCC patients with high USP8 expression.

The oncogenic role of ubiquitin specific peptidase (USP8) and its signaling pathways targeting for cancer therapeutics.

USP8 is a deubiquitinating enzyme in the family of ubiquitin-specific proteases (USPs) which can remove ubiquitin from the substrate and protect the substrate from degradation. The upregulated or mutated USP8 becomes hyperactivated and stabilizes numerous oncogenes or proto-oncogenes leading to cancer progression and survival by activating multiple signaling pathways. Moreover, USP8 inhibition is also important to overcome anticancer drug-resistant. This review is the first study to find, combine, analyze, and represent the multiple oncogenic signaling pathways with their downstream and upstream regulation activated or enhanced by USP8, which will help the researchers to find any therapeutic strategy for drug discovery by inhibiting or suppressing the multi-targeted USP8.

Discovery of novel USP8 inhibitors via Ubiquitin-Rho-110 fluorometric assay based high throughput screening.

USP8, one member of deubiquitinating enzymes (DUBs) families, maintains the ubiquitination level of EGFR and regulates the downstream signaling pathways. The deregulation of USP8 has been implicated in many human diseases, especially in cancer. Therefore, USP8 has been identified as a promising target for drug design. Herein, via high throughput screening based on Ubiquitin-rhodamine-110 (Ubiquitin-Rho-110) fluorometric activity assay, we discovered a novel inhibitor DC-U43. By structure optimization, DC-U43-10 reached a half-maximal inhibitory concentration (IC50) value of 2.6 ± 1.1 µM and exhibited 10-fold selectivity against USP7. The binding between DC-U43-10 and USP8 was validated by surface plasmon resonance (SPR) assay with a KD value of 10.5 ± 3.7 µM. It also inhibited the colony formation of H1975 cells. Hence, DC-U43-10 represents a kind of USP8 inhibitors with novel scaffold and has broad prospects for being a probe for USP8-related academic and clinical research.

The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma.

We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.

Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation.

Cushing's disease is primarily caused by autonomic hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. In Cushing's disease, mutations in the ubiquitin-specific protease 8 (USP8) have been detected. These mutations are associated with hyperactivation of USP8 that prevent epidermal growth factor receptor (EGFR) degradation. This leads to increased EGFR stability and results in the maintenance of EGFR signaling in Cushing's disease. USP8 inhibitors can suppress the growth of various tumors. In this study, the effects of a potent USP8 inhibitor, DUBs-IN-2, on ACTH production and cell proliferation were examined in mouse corticotroph tumor (AtT-20) cells. Proopiomelanocortin (Pomc) mRNA levels and ACTH levels were decreased in AtT-20 cells by DUBs-IN-2. Further, cell proliferation was inhibited, and apoptosis was induced by DUBs-IN-2. Transcript levels of pituitary tumor-transforming gene 1 (Pttg1), a pituitary tumor growth marker, were increased; and transcript levels of stress response growth arrest and DNA damage-inducible 45 (Gadd45ß) and Cdk5 and ABL enzyme substrate 1 (Cables1) mRNA levels were increased in response to the drug. Gadd45ß or Cables1 knockdown partially inhibited the DUBs-IN-2-induced decrease in cell proliferation, but not Pomc mRNA levels. Both GADD45ß and CABLES1 may be responsible, at least in part, for the USP8-induced suppression of corticotroph tumor cell proliferation. USP-8 may be a new treatment target in Cushing's disease.

miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling.

Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3'-untranslated region (3'-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3'-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.

The ESCRT-0 Protein HRS Interacts with the Human T Cell Leukemia Virus Type 2 Antisense Protein APH-2 and Suppresses Viral Replication.

The divergent clinical outcomes of human T cell leukemia virus type 1 (HTLV-1) and HTLV-2 infections have been attributed to functional differences in their antisense proteins. In contrast to HTLV-1 bZIP factor (HBZ), the role of the antisense protein of HTLV-2 (APH-2) in HTLV-2 infection is poorly understood. In previous studies, we identified the endosomal sorting complex required for transport 0 (ESCRT-0) subunit HRS as a novel interaction partner of APH-2 but not HBZ. HRS is a master regulator of endosomal protein sorting for lysosomal degradation and is hijacked by many viruses to promote replication. However, no studies to date have shown a link between HTLVs and HRS. In this study, we sought to characterize the interaction between HRS and APH-2 and to investigate the impact of HRS on the life cycle of HTLV-2. We confirmed a direct specific interaction between APH-2 and HRS and showed that the CC2 domain of HRS and the N-terminal domain of APH-2 mediate their interaction. We demonstrated that HRS recruits APH-2 to early endosomes, possibly furnishing an entry route into the endosomal/lysosomal pathway. We demonstrated that inhibition of this pathway using either bafilomycin or HRS overexpression substantially extends the half-life of APH-2 and stabilizes Tax2B expression levels. We found that HRS enhances Tax2B-mediated long terminal repeat (LTR) activation, while depletion of HRS enhances HTLV-2 production and release, indicating that HRS may have a negative impact on HTLV-2 replication. Overall, our study provides important new insights into the role of the ESCRT-0 HRS protein, and by extension the ESCRT machinery and the endosomal/lysosomal pathway, in HTLV-2 infection.IMPORTANCE While APH-2 is the only viral protein consistently expressed in infected carriers, its role in HTLV-2 infection is poorly understood. In this study, we characterized the interaction between the ESCRT-0 component HRS and APH-2 and explored the role of HRS in HTLV-2 replication. HRS is a master regulator of protein sorting for lysosomal degradation, a feature that is manipulated by several viruses to promote replication. Unexpectedly, we found that HRS targets APH-2 and possibly Tax2B for lysosomal degradation and has an overall negative impact on HTLV-2 replication and release. The negative impact of interactions between HTLV-2 regulatory proteins and HRS, and by extension the ESCRT machinery, may represent an important strategy used by HTLV-2 to limit virus production and to promote persistence, features that may contribute to the limited pathogenic potential of this infection.

HCRP-1 regulates EGFR-AKT-BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer.

Hepatocellular carcinoma-related protein-1 (HCRP-1), a subunit of mammalian endosomal sorting complex required for transport-I (ESCRT-I), is frequently downregulated in various kinds of malignant tumors. The role of HCRP-1 in colorectal cancer (CRC) remains unknown. We investigate the clinical value of HCRP-1 and its impact on anoikis in CRC. The negative expression of HCRP-1 was significantly correlated with tumor size (P = 0.033), PT status (P = 0.001), TNM stage (P = 0.039), and histological grade (P = 0.01). Univariate and multivariate analyses revealed that HCRP-1 was an independent prognostic factor for CRC (hazard ratio (HR) = 0.237, P < 0.001 for 5-year overall survival). In the in vitro assay, we found that HCRP-1 depletion resulted in cell anoikis resistance. Knockdown of HCRP-1 suppressed Bcl-2 interacting mediator of cell death (BIM) expression, with phosphorylation of AKT and p-FoxO3a, which was reversed by AKT siRNA or AKT inhibitor. Further analysis showed that loss of HCRP-1 obviously increased the activation of EGFR. Inhibition of EGFR blocked si-HCRP1-mediated phosphorylation of EGFR, AKT, FoxO3a, and BIM expression. Moreover, the in vivo results revealed that loss of HCRP-1 promoted cancer metastasis. Our findings implied that reduced HCRP-1 expression in CRC resulted in anoikis resistance and contributed to CRC metastasis and poor prognosis. These data may help design effective therapy targeting HCRP-1 pathway to control colon cancer growth and metastasis.

Potent Inhibition of Hepatitis E Virus Release by a Cyclic Peptide Inhibitor of the Interaction between Viral Open Reading Frame 3 Protein and Host Tumor Susceptibility Gene 101.

Hepatitis E virus (HEV) generally causes self-limiting acute viral hepatitis in normal individuals. It causes a more severe disease in immunocompromised persons and pregnant women. Due to the lack of an efficient cell culture system or animal model, the life cycle of the virus is understudied, few antiviral targets are known, and very few antiviral candidates against HEV infection have been identified. Inhibition of virus release is one possible antiviral development strategy, which limits the spread of the virus. Previous studies have demonstrated the essential role of the interaction between the PSAP motif of the viral open reading frame 3 protein (ORF3-PSAP) and the UEV domain of the host tumor susceptibility gene 101 (TSG101) protein (UEV-TSG101) in mediating the release of genotype 3 HEV. Cyclic peptide (CP) inhibitors of the interaction between the human immunodeficiency virus (HIV) gag-PTAP motif and UEV-TSG101 are known to block the release of HIV. Using a molecular dynamic simulation, we observed that both gag-PTAP and ORF3-PSAP motifs bind to the same site in UEV-TSG101 by hydrogen bonding. HIV-released inhibitory CPs also displayed binding to the same site in UEV-TSG101, indicating that they may compete with ORF3-PSAP or gag-PTAP for binding to UEV-TSG101. Two independent assays confirmed the ability of a cyclic peptide (CP11) to inhibit the ORF3-TSG101 interaction. CP11 treatment also reduced the release of both genotype 1 and genotype 3 HEV by approximately 90%, with a 50% inhibitory concentration (IC50) of 2 µM. Thus, CP11 appears to be an attractive candidate for further validation of its anti-HEV properties.IMPORTANCE There is no specific therapy against hepatitis E virus (HEV)-induced hepatic and nonhepatic health problems. Prevention of the release of the progeny viruses from infected cells is an attractive strategy to limit the spread of the virus. Interactions between the viral open reading frame 3 and the host tumor susceptibility gene 101 proteins have been shown to be essential for the release of genotype 3 HEV from infected cells. In this study, we have identified a cyclic peptide inhibitor of the above-mentioned interaction and demonstrate the efficiency of the inhibitor in preventing virus release from infected cells. Thus, our findings uncover the possibility of developing a specific antiviral agent against HEV by blocking its release from infected cells.

Resolving ESCRT-III Spirals at the Intercellular Bridge of Dividing Cells Using 3D STORM.

The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.

Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates ß-Cell Mitophagy.

Mitophagy is a cellular quality-control pathway, which is essential for elimination of unhealthy mitochondria. While mitophagy is critical to pancreatic ß-cell function, the posttranslational signals governing ß-cell mitochondrial turnover are unknown. Here, we report that ubiquitination is essential for the assembly of a mitophagy regulatory complex, comprised of the E3 ligase Nrdp1, the deubiquitinase enzyme USP8, and Clec16a, a mediator of ß-cell mitophagy with unclear function. We discover that the diabetes gene Clec16a encodes an E3 ligase, which promotes nondegradative ubiquitin conjugates to direct its mitophagy effectors and stabilize the Clec16a-Nrdp1-USP8 complex. Inhibition of the Clec16a pathway by the chemotherapeutic lenalidomide, a selective ubiquitin ligase inhibitor associated with new-onset diabetes, impairs ß-cell mitophagy, oxygen consumption, and insulin secretion. Indeed, patients treated with lenalidomide develop compromised ß-cell function. Moreover, the ß-cell Clec16a-Nrdp1-USP8 mitophagy complex is destabilized and dysfunctional after lenalidomide treatment as well as after glucolipotoxic stress. Thus, the Clec16a-Nrdp1-USP8 complex relies on ubiquitin signals to promote mitophagy and maintain mitochondrial quality control necessary for optimal ß-cell function.

A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.

Therapeutic Approaches for the Treatment of Epidermal Growth Factor Receptor Mutated Lung Cancer.

Lung cancer surfaces to be the predominant determinant of mortality worldwide constituting 13% and 19% of all new cancer cases and deaths related to cancer respectively. Molecular profiling has now become a regular trend in lung cancer to identify the driver mutations. Epidermal Growth Factor Receptor (EGFR) is the most regular driver mutation encountered in Non-Small Cell Lung Cancer (NSCLC). Targeted therapies are now available for the treatment of EGFR mutant NSCLC. EGFR mutation is more frequently expressed in adenocarcinoma than squamous cell carcinoma. This article presents a detailed molecular insight of the therapeutic approaches for the treatment of EGFR mutant lung cancer. The article delineates molecular mechanism of the drugs that are approved, the drugs that are in clinical trial and the drugs that have not entered a clinical trial but shows promising future in the treatment of EGFR mutant lung cancer. Furthermore, this article provides concise information on relevant combinational or monotherapy clinical trials that have been completed for various approaches.

TSG101: Tumor Susceptibility Gene 101 (tsg101) Product-Role in Therapy Against HIV/AIDS.

HIV infection presents a major community health hazard, partially because the HIV virus is capable of evading antiretroviral therapies. Most anti-HIV drugs were intended to target virus-encoded mechanisms; however, some host-encoded molecules comparatively execute a vital role in the life cycle of virus. Thus, these might be considered as target sites for antiviral agents. TSG101 is important among these antiviral therapies because, as a cytoplasmic molecule, it facilitates viral budding and release. In this review, HIV-infected cells have TSG101 on their surface and thus can be used in antibody-based therapies. The development of a monoclonal antibody CB8-2 lessens the assembly of viruses from infected cells. This mechanism represents the potential use of TSG101-directed antibodies to fight against AIDS.

Drug Discovery and Chemical Biology of Cancer Epigenetics.

Comprehensive whole-exome sequencing, DNA copy-number determination, and transcriptomic analyses of diverse cancers have greatly expanded our understanding of the biology of many tumor types. In addition to mutations in the common cell-of-origin specific driver mutations, these studies have also revealed a large number of loss-of-function and gain-of-function mutations in chromatin-modifying proteins (CMPs). This has revealed that epigenetic dysregulation is a common feature of most pediatric and adult cancers. Many specific and potent inhibitors have been developed for multiple CMP classes, which have assisted in elucidating the role of epigenetics as well as epigenetic vulnerabilities in these cancer types. Clinical trials with numerous CMP inhibitors are also currently in progress to evaluate the therapeutic potential of epigenetic inhibitors. In this review, we aim to provide a summary of genetic mutations in epigenetic genes and a review of CMP inhibitors suitable for preclinical studies or currently in clinical trials. Additionally, we highlight the CMPs for which potent inhibitors have not been developed and additional research focus should be dedicated.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.