Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

CD4 T cell-secreted IFN-γ in Sjögren's syndrome induces salivary gland epithelial cell ferroptosis.

BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disease that predominantly affects exocrine glands. Previous studies have demonstrated that upregulated interferon-gamma (IFN-γ) in SS triggers ferroptosis in salivary gland epithelial cells (SGECs), resulting in impaired salivary gland secretion. However, the immune cells responsible for secreting IFN-γ remain unclear. Therefore, this study conducted bioinformatics analysis and molecular validation to identify the origin of IFN-γ in SS salivary gland. METHODS: The 'limma' package in R software was utilized to identify differentially expressed genes (DEGs) in the human SS dataset. Subsequently, the identified DEGs were compared with the ferroptosis database and screened through Cytoscape to determine candidate genes. The cellular localization and expression patterns of candidate genes were further confirmed in the salivary gland single-cell RNA sequence (scRNA-seq) data set from healthy control and SS mice. Furthermore, in vitro and in vivo studies were performed to analyze the effect of CD4 T-secreted IFN-γ on SGECs' ferroptosis and functions. RESULTS: Upregulated TLR4, IFNG, and IL33 were screened as candidates ferroptosis ferroptosis-inducing genes in SS salivary glands. The association of IFNG and IL33 with CD4 T cells was established through immune infiltration analysis. The expression of IFN-γ on CD4 T cells was robustly higher compared with that of IL33 as evidenced by scRNA-seq and immunofluorescence co-localization. Subsequent experiments conducted on candidate genes consistently demonstrated the potent ability of IFN-γ to induce SGECs' ferroptosis and inhibit AQP5 expression. CONCLUSIONS: Our findings indicate that CD4 T cell-secreted IFN-γ in SS induces SGECs' ferroptosis and inhibits AQP5 expression.

Identification of microbial metabolites that accelerate the ubiquitin-dependent degradation of c-Myc.

Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library. The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained. In this study, we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp. RK19-A0402 strongly inhibits c-Myc transcriptional activity. After purification of the hit broth, we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A. Similar to oligomycin A, the hit compounds inhibited mitochondrial complex V. The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species (ROS), and the ROS activated GSK3α/ß that phosphorylated c-Myc for ubiquitination. This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.

Isolation and characterization of ß-transducin repeat-containing protein ligands screened using a high-throughput screening system.

ß-transducin repeat-containing protein (ß-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of ß-TrCP useful for the proteolysis targeting chimera (PROTAC) system using ß-TrCP in the future, we developed a high-throughput screening system for small molecule ß-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCFß-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of ß-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with ß-TrCP similar to those created by intrinsic target phosphopeptides of ß-TrCP. Our findings enhance our knowledge of useful small molecule ligands of ß-TrCP and the importance of residues that can be ligands of ß-TrCP.

Modes and Mechanisms of Salivary Gland Epithelial Cell Death in Sjogren's Syndrome.

Sjogren's syndrome is an autoimmune disease in middle and old-aged women with a dry mucosal surface, which is caused by the dysfunction of secretory glands, such as the oral cavity, eyeballs, and pharynx. Pathologically, Sjogren's syndrome are characterized by lymphocyte infiltration into the exocrine glands and epithelial cell destruction caused by autoantibodies Ro/SSA and La/SSB. At present, the exact pathogenesis of Sjogren's syndrome is unclear. Evidence suggests epithelial cell death and the subsequent dysfunction of salivary glands as the main causes of xerostomia. This review summarizes the modes of salivary gland epithelial cell death and their role in Sjogren's syndrome progression. The molecular mechanisms involved in salivary gland epithelial cell death during Sjogren's syndrome as potential leads to treating the disease are also discussed.

p-Coumaric acid attenuates the effects of Aß42 in vitro and in a Drosophila Alzheimer's disease model.

Alzheimer's disease (AD) is the most common neurodegenerative condition in civilizations worldwide. The distinctive occurrence of amyloid-beta (Aß) accumulation into insoluble fibrils is part of the disease pathophysiology with Aß42 being the most toxic and aggressive Aß species. The polyphenol, p-Coumaric acid (pCA), has been known to boost a number of therapeutic benefits. Here, pCA's potential to counteract the negative effects of Aß42 was investigated. First, pCA was confirmed to reduce Aß42 fibrillation using an in vitro activity assay. The compound was next examined on Aß42-exposed PC12 neuronal cells and was found to significantly decrease Aß42-induced cell mortality. pCA was then examined using an AD Drosophila melanogaster model. Feeding of pCA partially reversed the rough eye phenotype, significantly lengthened AD Drosophila's lifespan, and significantly enhanced the majority of the AD Drosophila's mobility in a sex-dependent manner. The findings of this study suggest that pCA may have therapeutic benefits for AD.

Identification of antimycin A as a c-Myc degradation accelerator via high-throughput screening.

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.

Interferon-γ induces salivary gland epithelial cell ferroptosis in Sjogren's syndrome via JAK/STAT1-mediated inhibition of system Xc.

The elevated level of interferon-γ (IFN-γ) in Sjogren's syndrome (SS) triggers salivary gland epithelial cells (SGEC) death. However, the underlying mechanisms of IFN-γ-induced SGEC death modes are still not fully elucidated. We found that IFN-γ triggers SGEC ferroptosis via Janus kinase/signal transducer and activator of transcription 1 (JAK/STAT1)-mediated inhibition of cystine-glutamate exchanger (System Xc-). Transcriptome analysis revealed that ferroptosis-related markers are differentially expressed in SS human and mouse salivary glands with distinct upregulation of IFN-γ and downregulation of glutathione peroxidase 4 (GPX4) and aquaporin 5 (AQP5). Inducing ferroptosis or IFN-γ treatment in the Institute of cancer research (ICR) mice aggravated and inhibition of ferroptosis or IFN-γ signaling in SS model non-obese diabetic (NOD) mice alleviated ferroptosis in the salivary gland and SS symptoms. IFN-γ activated STAT1 phosphorylation and downregulated system Xc- components solute carrier family 3 member 2 (SLC3A2), glutathione, and GPX4 thereby triggering ferroptosis in SGEC. JAK or STAT1 inhibition in SGEC rescued IFN-γ-downregulated SLC3A2 and GPX4 as well as IFN-γ-induced cell death. Our results indicate the role of ferroptosis in SS-related death of SGEC and SS pathogenicity.

Dental pulp stem cell-derived exosomes revitalize salivary gland epithelial cell function in NOD mice via the GPER-mediated cAMP/PKA/CREB signaling pathway.

BACKGROUND: Restoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet. METHODS: DPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis. RESULTS: DPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER. CONCLUSIONS: Our results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.

Role of estrogen receptor signaling pathway-related genes in diffuse large B-cell lymphoma and identification of key targets via integrated bioinformatics analysis and experimental validation.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignancy. Epidemiologically, the incidence of DLBCL is higher in men, and the female sex is a favorable prognostic factor, which can be explained by estrogen. This study aimed to explore the potential targets of the estrogen receptor (ER) signaling pathway and provide a meaningful way to treat DLBCL patients. Datasets were obtained from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs). Representative gene sets estrogen receptor pathways, and growth regulatory pathways were identified based on Gene Set Enrichment Analysis (GSEA) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for function and pathway analysis. STRING and Cytoscape were used to construct the interaction network, and the MCODE plug-in performed the module analysis. GEPIA, TCGA, and LOGpc databases were used for expression and predictive analysis. The Human Protein Atlas (HPA) database was used to analyze the protein expression levels, cBioPortal was used to explore genetic alterations, and ROC analysis and prognostic assessment were used to predict the diagnostic value of genes. Finally, BJAB cells were treated with ER inhibitor fulvestrant and specific shRNA, and the expression of hub genes was verified by RT-qPCR. We identified 81 overlapping DEGs and CDC6, CDC20, KIF20A, STIL, and TOP2A as novel biomarkers affecting the prognosis of DLBCL. In addition, the STAT and KRAS pathways are considered potential growth regulatory pathways. These results hold promise for new avenues for the treatment of DLBCL patients.

Lonidamine and domperidone inhibit expansion of transformed cell areas by modulating motility of surrounding nontransformed cells.

Cancer cells intrinsically proliferate in an autonomous manner; however, the expansion of cancer cell areas in a tissue is known to be regulated by surrounding nontransformed cells. Whether these nontransformed cells can be targeted to control the spread of cancer cells is not understood. In this study, we established a system to evaluate the cancer-inhibitory activity of surrounding nontransformed cells and screened chemical compounds that could induce this activity. Our findings revealed that lonidamine (LND) and domperidone (DPD) inhibited expansion of oncogenic foci of KRASG12D-expressing transformed cells, whereas they did not inhibit the proliferation of monocultured KRASG12D-expressing cells. Live imaging revealed that LND and DPD suppressed the movement of nontransformed cells away from the attaching cancer cells. Moreover, we determined that LND and DPD promoted stress fiber formation, and the dominant-negative mutant of a small GTPase RhoA relieved the suppression of focus expansion, suggesting that RhoA-mediated stress fiber formation is involved in the inhibition of the movement of nontransformed cells and focus expansion. In conclusion, we suggest that elucidation of the mechanism of action of LND and DPD may lead to the development of a new type of drug that could induce the anticancer activity of surrounding nontransformed cells.

Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation.

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

ZMIZ2 promotes the development of triple-receptor negative breast cancer.

BACKGROUND: Triple-receptor negative breast cancer (TNBC) is an aggressive breast tumor subtype that generally has a poor prognosis. This study aimed to investigate the role and regulatory mechanisms of Zinc finger MIZ-type containing 2 (ZMIZ2) in relation to TNBC. METHODS: Based on data from The Cancer Genome Atlas (TCGA), the expression of ZMIZ2 in different subtypes and its correlation with androgen receptor (AR) were analyzed, and a regulatory mechanism network was constructed. The expression and prognostic value of ZMIZ2 in clinical TNBC tissue samples were also investigated. Furthermore, in vitro studies were conducted to investigate the effects of ZMIZ2 knockdown on the malignant behaviors of TNBC cells and target gene expression. RESULTS: Based on TCGA data, ZMIZ2 was found to be significantly upregulated in TNBC tissues and its expression was negatively correlated with AR expression. Key relationships, such as the ZMIZ2-CCL5, ZMIZ2/AR-MCM3, ZMIZ2/AR-E2F4, and the ZMIZ2/AR-DHX38 were identified, which were enriched in NOD-like receptor signaling pathway/toll-like receptor signaling pathway, DNA replication, cell cycle, and spliceosome, respectively. Moreover, ZMIZ2 was upregulated in clinical breast cancer tissues and its high expression was correlated with the poor prognosis of TNBC patients. Furthermore, ZMIZ2 expression was increased in breast cancer cells, and a knockdown of ZMIZ2 inhibited MDA-MB-231 cell proliferation, migration, and invasion, induced cell cycle arrest in the G1 phase, and promoted cell apoptosis. Furthermore, ZMIZ2 knockdown inhibited the mRNA and protein expression of CCL5, MCM3, E2F4, and DHX38. CONCLUSION: Our findings reveal that ZMIZ2 is upregulated in TNBC tissues and is associated with its poor prognosis. ZMIZ2 may promote TNBC progression by promoting the expression of its target genes and affecting the corresponding pathways. Consequently, ZMIZ2 may serve as a promising target for future TNBC treatments.

Identification of a Small Molecule That Enhances Ferroptosis via Inhibition of Ferroptosis Suppressor Protein 1 (FSP1).

Glutathione peroxidase 4 (GPX4) is an intracellular enzyme that oxidizes glutathione while reducing lipid peroxides and is a promising target for cancer therapy. To date, several GPX4 inhibitors have been reported to exhibit cytotoxicity against cancer cells. However, some cancer cells are less sensitive to the known GPX4 inhibitors. This study aimed to explore compounds showing synergistic effects with GPX4 inhibitors. We screened a chemical library and identified a compound named NPD4928, whose cytotoxicity was enhanced in the presence of a GPX4 inhibitor. Furthermore, we identified ferroptosis suppressor protein 1 as its target protein. The results indicate that NPD4928 enhanced the sensitivity of various cancer cells to GPX4 inhibitors, suggesting that the combination might have therapeutic potential via the induction of ferroptosis.

Ethyl caffeate ameliorated amyloid-beta42 protein-associated toxicity in PC12 cells and Drosophila melanogaster.

AIM: Alzheimer's disease (AD) is the most pervasive neurodegenerative disorder in societies globally. Till now, the mechanism behind this disease is still equivocal. Amyloid-beta42 protein (Aß42), the most toxic and aggressive Aß species, is the main focus of this study. The naturally occurring ethyl caffeate (EC) is associated with various medicinal properties. Here, EC was tested for its protective properties against Aß42's toxic effects. METHODS: As treatment of Aß42 has been shown to cause neuronal cell death, EC was first screened with Aß42-incubated PC12 neuronal cells. Next, the compound was tested on the Drosophila melanogaster AD model using the rough eye phenotype assay, lifespan assay and negative geotaxis assay. RESULTS: EC ameliorated PC12 cells from cell death linked to Aß42 exposure. Using Drosophila expressing human Aß42, feeding of EC was able to partially rescue the rough eye phenotype, lengthen the lifespan of AD Drosophila and enhanced the mobility of middle-aged AD Drosophila. CONCLUSION: Overall, the results of this study showed that EC might possess therapeutic properties for AD. Geriatr Gerontol Int 2021; 21: 1125-1130.

Alleviatory effects of Danshen, Salvianolic acid A and Salvianolic acid B on PC12 neuronal cells and Drosophila melanogaster model of Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen water extract (DWE), obtained from the Salvia miltiorrhiza Bunge (Family Lamiaceae) root, is usually employed in Chinese traditional medicine as treatment to cardiovascular ailments and cerebrovascular diseases. Intriguingly, the extract was also found to contain vast beneficial properties in Alzheimer's disease (AD) treatment. AIM OF THE STUDY: Alzheimer's disease is the most significant type of neurodegenerative disorder plaguing societies globally. Its pathogenesis encompasses the hallmark aggregation of amyloid-beta (Aß). Of all the Aß oligomers formed in the brain, Aß42 is the most toxic and aggressive. Despite this, the mechanism behind this disease remains elusive. In this study, DWE, and its major components, Salvianolic acid A (SalA) and Salvianolic acid B (SalB) were tested for their abilities to attenuate Aß42's toxic effects. METHODS: The composition of DWE was determined via Ultra-Performance Liquid Chromatography (UPLC). DWE, SalA and SalB were first verified for their capability to diminish Aß42 fibrillation using an in vitro activity assay. Since Aß42 aggregation results in neuronal degeneration, the potential Aß42 inhibitors were next evaluated on Aß42-exposed PC12 neuronal cells. The Drosophila melanogaster AD model was then employed to determine the effects of DWE, SalA and SalB. RESULTS: DWE, SalA and SalB were shown to be able to reduce fibrillation of Aß42. When tested on PC12 neuronal cells, DWE, SalA and SalB ameliorated cells from cell death associated with Aß42 exposure. Next, DWE and its components were tested on the Drosophila melanogaster AD model and their rescue effects were further characterized. The UPLC analysis showed that SalA and SalB were present in the brains and bodies of Drosophila after DWE feeding. When human Aß42 was expressed, the AD Drosophila exhibited degenerated eye structures known as the rough eye phenotype (REP), reduced lifespan and deteriorated locomotor ability. Administration of DWE, SalA and SalB partially reverted the REP, increased the age of AD Drosophila and improved most of the mobility of AD Drosophila. CONCLUSION: Collectively, DWE and its components may have therapeutic potential for AD patients and possibly other forms of brain diseases.

Discovery of a σ1 receptor antagonist by combination of unbiased cell painting and thermal proteome profiling.

Phenotypic screening for bioactive small molecules is typically combined with affinity-based chemical proteomics to uncover the respective molecular targets. However, such assays and the explored bioactivity are biased toward the monitored phenotype, and target identification often requires chemical derivatization of the hit compound. In contrast, unbiased cellular profiling approaches record hundreds of parameters upon compound perturbation to map bioactivity in a broader biological context and may link a profile to the molecular target or mode of action. Herein we report the discovery of the diaminopyrimidine DP68 as a Sigma 1 (σ1) receptor antagonist by combining morphological profiling using the Cell Painting assay and thermal proteome profiling. Our results highlight that integration of complementary profiling approaches may enable both detection of bioactivity and target identification for small molecules.

Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry.

Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several other methods used for drug-target identification, such as biochemical fractionation or radioactive ligand binding assay, affinity purification is widely used for its straight-forward and easy approach. To identify the target protein of an inhibitor of cancer cell-accelerated fibroblast migration, we prepared the inhibitor-conjugated beads by photo-crosslinking. Proteins were pulled down from cell lysates by the compound beads and separated by SDS-PAGE, and a specifically pulled down protein was cut out, trypsin-digested, analyzed using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) and identified by peptide mass fingerprinting (PMF) method. Since the photo-crosslinking enables the immobilization of ligands on an affinity matrix in a functional group-independent manner, we do not have to determine the functional group of the compound to conjugate the matrix. In addition, as compared to other MS techniques such as electrospray ionization, MALDI offers a less complex sample preparation procedure and higher sensitivity, and thus is better suited for the rapid identification of proteins isolated by gel electrophoresis.

SUMO3 modification by PIAS1 modulates androgen receptor cellular distribution and stability.

BACKGROUND: Abnormal reactivation of androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) mainly results from overexpression and down-regulation of AR. Sumoylation of AR can influence its function. However, regulation of AR sumoylation by SUMO E3 ligases PIASs to modify AR distribution and stability are not well understood. METHODS: We assessed the potential effect of SUMO3 modification on AR intracellular localization by immunostaining in AR-negative prostate cancer DU145 cells, and detected the effect of PIAS1/SUMO3 overexpression on AR sumoylation related degradation. Then we characterized AR sumoylation sites involved modified by SUMO3, and the key residue of PIAS1 involved in itself sumoylation and further mediated AR sumoylation (sumo3-conjugated), translocation and degradation. Finally we detected the recognition of PIAS1 (sumoylation ligase) to MDM2, a ubiquin ligase mediated AR degradation. RESULTS: We demonstrate that SUMO E3 ligase PIAS1, along with SUMO3, mediates AR cytosolic translocation and subsequent degradation via a ubiquitin-proteasome pathway. Although AR sumoylation occurs prior to ubiquitination, the SUMO-acceptor lysine 386 on AR, together with ubiquitin-acceptor lysine 845, contribute to PIAS1/SUMO3-induced AR nuclear export, ubiquitination and subsequent degradation. Moreover, PIAS1 itself is modified by SUMO3 overexpression, and mutation of SUMO-acceptor lysine 117 on PIAS1 can impair AR cytoplasmic distribution, demonstrating the essential role of sumoylated PIAS1 in AR translocation. We further determine that sumoylated PIAS1 interacts with AR lysine 386 and 845 to form a binary complex. Consistent with the effect on AR distribution, SUMO3 modification of PIAS1 is also required for AR ubiquitination and degradation by recruiting ubiquitin E3 ligase MDM2. CONCLUSION: Taken together, SUMO3 modification of PIAS1 modulates AR cellular distribution and stability. Our study provided the evidence the crosstalk between AR sumoylation and ubquitination mediated by PIAS1 and SUMO3.