ABSTRACT
Autophagy is a highly conserved cellular homeostasis maintenance mechanism in eukaryotes. Microtubule-associated protein light chain 3 (LC3) plays a crucial role in autophagy. It has multiple pairs of protein-protein interactions (PPIs) with other proteins, and these PPIs have an effect on the regulation of autophagosome formation and the recruitment of autophagic substrates. In our previous work, a small molecule covalent inhibitor DC-LC3in-D5 which could inhibit LC3A/B PPIs was identified, but a detailed study of structure-activity relationships (SARs) was lacking. Herein, a new molecule LC3in-C42 was discovered utilizing the hybridization of advantageous fragments, whose potency (IC50 = 7.6 nM) had been greatly improved compared with that of DC-LC3in-D5. LC3in-C42 inhibits autophagy at the cellular level and its efficacy far exceeds that of DC-LC3in-D5. Thus far, LC3in-C42 stands as the most potent LC3A/B small molecule inhibitor. LC3in-C42 could serve as a powerful tool for LC3A/B protein and autophagy research.
Subject(s)
Autophagy , Microtubule-Associated Proteins , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/antagonists & inhibitors , Humans , Structure-Activity Relationship , Autophagy/drug effects , Drug Design , Molecular Structure , Drug DiscoveryABSTRACT
Autophagy is a non-selective action in which cells degrade parts of themselves, reusing degraded cellular components. Among autophagy-related gene (ATG) family members, ATG4 proteins play crucial roles in the microtubule-associated protein 1 light chain 3 (LC3) phosphatidylethanolamine (PE) system which is essential for autophagosome maturation. Although autophagy has been shown to be involved in osteoclastic bone resorption, the role of ATG4/LC3 in bone resorption remains unclear. When mouse bone marrow cells were treated with various concentrations of NSC185058 (NSC), a specific inhibitor of ATG4B, 1 h prior to treatment with receptor activator of NF-κB ligand (RANKL) in the presence of macrophage colony stimulating factor (M-CSF), NSC inhibited osteoclastogenesis in a dose-dependent manner. Addition of NSC in the late stages of osteoclast differentiation suppressed multinucleation and reduced the expression of markers for mature osteoclasts such as Dc-stamp, Mmp9, and Ctsk. NSC also suppressed actin ring formation and pit formation in mature osteoclasts. When a periodontitis model involving eight-week-old male mice in which the right maxillary second molar had been ligated with silk thread was injected with or without NSC, alveolar bone resorption was suppressed by a decrease in the number of osteoclasts in the NSC-treated group. These results suggest that LC3 is important for the maturation of osteoclasts and that LC3 inhibition is a new therapeutic strategy for periodontal disease.
Subject(s)
Cell Differentiation , Osteoclasts , Animals , Male , Mice , Actins/metabolism , Alveolar Bone Loss , Ligands , Macrophage Colony-Stimulating Factor/pharmacology , Macrophage Colony-Stimulating Factor/metabolism , Matrix Metalloproteinase 9/metabolism , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/metabolism , Osteoclasts/metabolism , Phosphatidylethanolamines/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Silk , Bone Resorption/genetics , Bone Resorption/metabolism , Cell Differentiation/genetics , Autophagy-Related Proteins/antagonists & inhibitors , Autophagy-Related Proteins/metabolism , Cysteine Endopeptidases/metabolismABSTRACT
In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.
Subject(s)
Autophagosomes/drug effects , Autophagy/drug effects , Dendritic Cells/drug effects , Phagocytosis/drug effects , Proteasome Endopeptidase Complex/drug effects , Autophagosomes/metabolism , Autophagy/genetics , Cell Differentiation , Chloroquine/pharmacology , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/genetics , Class III Phosphatidylinositol 3-Kinases/metabolism , Dendritic Cells/cytology , Dendritic Cells/metabolism , Endosomes/drug effects , Endosomes/metabolism , Gene Expression Regulation , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Macrolides/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Monocytes/cytology , Monocytes/metabolism , Phagocytosis/genetics , Phagosomes/drug effects , Phagosomes/metabolism , Primary Cell Culture , Proteasome Endopeptidase Complex/metabolism , Pyridines/pharmacology , Pyrimidinones/pharmacology , Thiophenes/pharmacology , Zymosan/metabolismABSTRACT
Fibroblast growth factor receptor 3-transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3), which has been identified in many cancers such as glioblastoma and bladder cancer, is a potent oncogenic fusion protein that induces constitutive activation of FGFR signaling, resulting in uncontrolled cell proliferation. Although several tyrosine kinase inhibitors against FGFR are currently under development, resistance to such types of inhibitors in patients has become a concern. In this study, a chimeric molecule SNIPER(TACC3)-11 (5a) was developed and found to reduce FGFR3-TACC3 levels effectively. Compound 5a conjugated KHS108 (a TACC3 ligand) to an LCL161 derivative (11) (an inhibitor of apoptosis protein [IAP] ligand) with a PEG linker (n = 2). Mechanistical analysis showed that cellular IAP1 was required for the reduction of FGFR3-TACC3 levels. Consistent with the decrease in FGFR3-TACC3 levels, compound 5a suppressed the growth of FGFR3-TACC3 positive cells. Thus, compound 5a is a candidate therapeutic with a novel drug modality against cancers that exhibit FGFR3-TACC3-dependent proliferation and exerts pharmacological effects distinct from FGFR3 kinase inhibitors because it lacks substructures crucial for kinase inhibition.
Subject(s)
Antineoplastic Agents , Drug Development , Microtubule-Associated Proteins , Receptor, Fibroblast Growth Factor, Type 3 , Humans , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/metabolism , Molecular Structure , Receptor, Fibroblast Growth Factor, Type 3/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Structure-Activity RelationshipABSTRACT
BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , SARS-CoV-2/genetics , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/pathology , COVID-19/virology , Chlorocebus aethiops , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Editing/methods , HEK293 Cells , Host-Pathogen Interactions/drug effects , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Proteins/antagonists & inhibitors , Proteins/genetics , Proteins/metabolism , RNA Interference , RNA, Guide, Kinetoplastida/metabolism , RNA, Small Interfering/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Vero Cells , Virus Replication/genetics , COVID-19 Drug TreatmentABSTRACT
The centrosome is a special organelle in human cells and an organizing unit for microtubules and signaling molecules. In addition, the centrosome is tightly restricted during the cell cycle and forms the basal body of the cilia in ciliated cells. Centrosome abnormality is frequently observed in malignant tumors. The dysregulation of centrosome-associated proteins leads to multipolar mitosis, aneuploidy, and nondirected cell migration, and therefore promotes cancer progression. The overduplication of primary centrosome and the accumulation of chromosome, comprise the majority cause of chromosomal mis-segregation in cancer cells. This review discusses the structure and function of the centrosome and the role of its associated proteins in the progression of solid tumors. We summarized the effects of centrosome amplification abnormalities and other centrosome-related phenotypes on tumors. The mechanism of the delineation of centrosome amplification with tumor malignancy remains to be decided. A better understanding of centrosome abnormality in tumorigenesis may be useful to screen novel therapeutic strategies for the treatment of solid tumors.
Subject(s)
Cell Cycle Proteins/metabolism , Centrosome/metabolism , Microtubule-Associated Proteins/metabolism , Neoplasms/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Centrosome/drug effects , Centrosome/pathology , Gene Expression Regulation, Neoplastic , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Signal TransductionABSTRACT
The dynamic equilibrium of tubulin-microtubule is an essential aspect of cell survivality. Modulation of this dynamics has become an important target for the cancer drug development. Tubulin exists in the alpha-beta dimer form which polymerizes to form microtubule and further depolymerizes back to tubulin dimer. The microtubule plays an essential role in mitosis and cell multiplication. Antitubulin drugs disturb the microtubule dynamics which is essentially required for DNA segregation and cell division during mitosis so killing the cancerous cells. Microtubule Associated Proteins (MAPs) interact with cellular cytoskeletal microtubules. MAPs bind to the either polymerized or depolymerized tubulin dimers within the cell and mostly causing stabilization of microtubules. Some of the tubulin binding drugs are in clinical use and others in clinical trial. MAPs inhibitors are also in clinical trial. Post-translational modification of lysine-40 either in histone or in alpha tubulin has an important role in gene expression and is balanced between histone deacetylases (HDACs) and histone acetyltransferases (HATs). HDAC inhibitors have the anticancer properties to form a drug for the treatment of cancer. They act by inducing cell cycle arrest and cell death. Some of the HDAC inhibitors are approved to be used as anticancer drug while others are under different phases of clinical trial. The present review updates on various MAPs, their role in cancer progression, MAPs inhibitors and their future prospects.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Development , Histone Deacetylase Inhibitors/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Tubulin Modulators/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/metabolism , Humans , Microtubule-Associated Proteins/metabolism , Molecular Structure , Tubulin/metabolism , Tubulin Modulators/chemical synthesis , Tubulin Modulators/chemistryABSTRACT
Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert antiinflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVAinduced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)5, IL13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVAinduced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagyrelated gene 4microtubuleassociated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an antiinflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Asthma/etiology , Autophagy/drug effects , Drugs, Chinese Herbal/pharmacology , Sapogenins/pharmacology , Animals , Asthma/drug therapy , Asthma/metabolism , Autophagy-Related Proteins/antagonists & inhibitors , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Biomarkers , Biopsy , Bronchial Hyperreactivity/drug therapy , Bronchial Hyperreactivity/etiology , Bronchial Hyperreactivity/metabolism , Bronchoalveolar Lavage Fluid , Cytokines/metabolism , Disease Management , Disease Models, Animal , Disease Susceptibility , Female , Immunoglobulin E/blood , Immunoglobulin E/immunology , Immunohistochemistry , Inflammation Mediators/metabolism , Mice , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Sapogenins/chemistry , Structure-Activity RelationshipABSTRACT
Cellular autophagy is an intracellular degradation pathway, which transports damaged, deformed, senescent or non-functional proteins and organelles to lysosome for digestion and degradation. Cellular autophagy is deeply evolutionarily conservedfromyeasttomammaliancells, and many homologous proteins of the autophahgy regulators are found in several species. This physiological process maintains the steady state of cells. Furtheremore, autophagy dysfunction is closely related to various diseases, such as neurodegenerative diseases, inflammation-related diseases, cardiovascular diseases, metabolic diseases, etc. The LC3 and p62 protein protein interaction (PPI) promotes the formation of autophagosomes and delivers polyubiquitinated "cargoes" to autophagic degradation. Therefore, LC3-p62 PPI plays an integral role in the formation of autophagosomes and effectively inhibits autophagy. However, there are still few studies on the LC3-p62 PPI inhibitors for its unclear molecular mechanism. Furthermore, most of these inhibitors are macromolecules with poorly active, and small molecules are particularly scarce. In this article, the computation method was used to identify the hot spot and design peptides as the binder of LC3-p62 PPI. Findings from this work provide a reference for the follow-up research of discovering small molecule inhibitors targeting LC3-p62 PPI.
Subject(s)
Drug Design , Microtubule-Associated Proteins/antagonists & inhibitors , Peptides/pharmacology , RNA-Binding Proteins/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Models, Molecular , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistry , Protein Binding , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.
Subject(s)
Biophysical Phenomena , Cell Cycle Proteins/chemistry , Epigenesis, Genetic , Microtubule-Associated Proteins/chemistry , Phosphoproteins/chemistry , Protein Conformation , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/ultrastructure , Entropy , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/ultrastructure , Molecular Docking Simulation , Molecular Dynamics Simulation , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/ultrastructure , Protein Binding , Structure-Activity RelationshipABSTRACT
Emerging evidence suggests that microRNAs (miRNAs) participate in hepatocellular carcinoma (HCC) progression. Nevertheless, the mechanism of miR-7-5p in HCC cells has not been researched. In the research, the underlying biological function of miR-7-5p and SPC24 in HCC was explored. qRT-PCR was performed to measure the miR-7-5p and SPC24 level in HCC tissues and cells. The effect of miR-7-5p on HCC progression was detected by performing CCK-8, BrdU, and transwell assay. The relationship between miR-7-5p and SPC24 was determined using luciferase and RNA pull-down assays. Our findings showed that miR-7-5p was downregulated in HCC whereas SPC24 was upregulated in HCC. It was also showed that miR-7-5p upregulation restricted malignant behaviors of HCC cells, but this inhibitory effect of miR-7-5p could be relieved by its target gene SPC24. In conclusion, this research suggested that by inhibiting SPC24, miR-7-5p could act as a tumor inhibitory factor in HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MicroRNAs/genetics , Microtubule-Associated Proteins/antagonists & inhibitors , Apoptosis/physiology , Carcinoma, Hepatocellular/genetics , Cell Line , Cell Line, Tumor , Cell Movement/physiology , Cell Proliferation/physiology , Computational Biology/methods , Humans , Liver Neoplasms/genetics , MicroRNAs/metabolism , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Signal TransductionABSTRACT
Acetyl-L-carnitine has been shown to exert neuroprotection against neurodegenerative diseases. The present study was performed to evaluate neuroprotection effects of acetyl-L-carnitine against lipopolysaccharide (LPS) -induced neuroinflammation and clarify possible mechanisms. A single dose (500 µg/kg) of LPS was intraperitoneally injected to rats to induce model. The animals were intraperitoneally treated with different doses of acetyl-L-carnitine (30, 60, and 100) for 6 days. Y-maze task, single-trial passive avoidance and novel object recognition tests were used to evaluate memory impairments. ELISA assay was used to evaluate the expression of TLR4/NFκB, autophagic and oxidative stress markers. Our result showed that intraperitoneal injection of LPS resulted in initiation of neuroinflammation by activation of TLR4/NFκB, suppression of autophagic markers such as LC3 II/ LC3 I ratio and becline-1, and excessive production of ROS and MDA. Intraperitoneal administration of acetyl-L-carnitine contributed to neuroprotection against LPS -induced neuroinflammation by suppression of TLR4/NFκB pathway, restoring activity of autophagy and inhibition of oxidative stress. Collectively, our findings show that acetyl-L-carnitine attenuated LPS-induced neuroinflammation by targeting TLR4/NFκB pathway, autophagy and oxidative stress.
Subject(s)
Acetylcarnitine/pharmacology , Anti-Inflammatory Agents/pharmacology , Autophagy/drug effects , Lipopolysaccharides , NF-kappa B/drug effects , Neuroinflammatory Diseases/prevention & control , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Toll-Like Receptor 4/drug effects , Animals , Beclin-1/antagonists & inhibitors , Injections, Intraperitoneal , Male , Microtubule-Associated Proteins/antagonists & inhibitors , Neuroinflammatory Diseases/chemically induced , Neuroinflammatory Diseases/psychology , Psychomotor Performance/drug effects , Rats , Rats, WistarABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.
Subject(s)
COVID-19/metabolism , COVID-19/virology , Calcium Signaling/physiology , Microtubule-Associated Proteins/metabolism , NADP/analogs & derivatives , SARS-CoV-2/physiology , Affinity Labels , Animals , Calcium Channels/metabolism , Carrier Proteins/metabolism , Click Chemistry/methods , Gene Knockdown Techniques , HEK293 Cells , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Second Messenger Systems/physiology , Transcriptome , Virus InternalizationABSTRACT
As a newly emerged technology, PROTAC (proteolysis targeting chimera) is a promising therapeutic strategy for varieties of diseases. Unlike small molecule inhibitors, PROTACs catalytically induce target proteins degradation, including currently "undruggable" target proteins. In addition, PROTACs can be a potentially successful strategy to overcome drug resistance. IAPs can inhibit apoptosis by inhibiting caspase, and also exhibits the activity of E3 ubiquitin ligase. Specific and nongenetic IAP-based protein erasers (SNIPERs) are hybrid molecules that designed based on IAPs, and used to degrade the target proteins closely associated with diseases. Their structures consist of three parts, including target protein ligand, E3 ligase ligand and the linker between them. SNIPERs (PROTACs) degrade diseases-associated proteins through human inherent ubiquitin-proteasome system. So far, many SNIPERs have been developed to treat diseases that difficult to handle by traditional methods, such as radiotherapy, chemotherapy and small molecule inhibitors, and showed promising prospects in application. In this paper, the recent advances of SNIPERs were summarized, and the chances and challenges associated with this area were also highlighted.
Subject(s)
Inhibitor of Apoptosis Proteins/metabolism , Ligands , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , Humans , Huntingtin Protein/antagonists & inhibitors , Huntingtin Protein/metabolism , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Receptor, Notch1/antagonists & inhibitors , Receptor, Notch1/metabolism , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism , Receptors, Estrogen/metabolismABSTRACT
Prostate cancer with high Gleason grade is prone to metastasis, which is one of the factors that seriously threaten the survival of patients, and it is also a treatment difficulty. In this study, we first revealed the potential connection between TPX2 and prostate cancer metastasis. We found that TPX2 is highly expressed in high-grade prostate cancer and is significantly related to poor prognosis. Depletion of TPX2 can significantly inhibit cell activity and migration, and in vivo experiments show that knockdown of TPX2 can significantly inhibit tumor growth. In terms of mechanism, we found that knocking down TPX2 can inhibit the expression of CDK1, repress the phosphorylation of ERK/GSK3ß/SNAIL signaling pathway, and thereby inhibit tumor epithelial-mesenchymal transition. Subsequently, we found that after rescuing TPX2, all related proteins and phenotype changes were restored, and this effect can be inhibited by CDK1 inhibitor, RO-3306. Our findings suggest the potential of TPX2 as an important target in anti-tumor metastasis therapy, which is conducive to precision medicine for prostate cancer.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Epithelial-Mesenchymal Transition , Glycogen Synthase Kinase 3 beta/metabolism , MAP Kinase Signaling System , Microtubule-Associated Proteins/metabolism , Prostatic Neoplasms/metabolism , Snail Family Transcription Factors/metabolism , Animals , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Epithelial-Mesenchymal Transition/drug effects , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Nude , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Neoplasm Metastasis/drug therapy , Phosphorylation/drug effects , Prognosis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Quinolines/pharmacology , Thiazoles/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Collapsin response mediator protein 5 (CRMP5), a member of the CRMP family, is expressed in the brain, particularly in the hippocampus, an area of the brain that can modulate stress responses. Social stress has a well-known detrimental effect on health and can lead to depression, but not all individuals are equally sensitive to stress. To date, researchers have not conclusively determined how social stress increases the susceptibility of the brain to depression. Here, we used the chronic social defeat stress (CSDS) model and observed higher hippocampal CRMP5 expression in stress-susceptible (SS) mice than in control and stress-resilient (RES) mice. A negative correlation was observed between the expression levels of CRMP5 and the social interaction (SI) ratio. Reduced hippocampal CRMP5 expression increased the SI ratio in SS mice, whereas CRMP5 overexpression was sufficient to induce social avoidance behaviors in control mice following exposure to subthreshold social stress induced by lentivirus-based overexpression and inducible tetracycline-on strategies to upregulate CRMP5. Interestingly, increased CRMP5 expression in SS and lenti-CRMP5-treated mice also caused serum corticosterone concentrations to increase. These findings improve our understanding of the potential mechanism by which CRMP5 triggers susceptibility to social stress, and they support the further development of therapeutic agents for the treatment of stress disorders in humans.
Subject(s)
Hydrolases/biosynthesis , Hydrolases/genetics , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Social Defeat , Stress, Psychological/genetics , Stress, Psychological/metabolism , Animals , Gene Knockdown Techniques/methods , HEK293 Cells , Hippocampus/metabolism , Humans , Hydrolases/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/antagonists & inhibitors , Stress, Psychological/psychologyABSTRACT
Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes-an intracellular bacterial pathogen-suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host-microbial interactions.
Subject(s)
Calcium Signaling , Listeria monocytogenes/physiology , Microtubule-Associated Proteins/antagonists & inhibitors , Mitochondria/metabolism , Phagocytosis , Acetyl Coenzyme A/metabolism , Acetylation , Animals , Autophagy-Related Proteins/metabolism , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Host-Pathogen Interactions , Humans , Listeria monocytogenes/growth & development , Listeria monocytogenes/metabolism , Macrophages/metabolism , Macrophages/microbiology , Mice , Microtubule-Associated Proteins/metabolism , Mutation , NADPH Oxidases/metabolismABSTRACT
The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Survival , Drosophila Proteins/antagonists & inhibitors , Female , Inhibitor of Apoptosis Proteins/metabolism , Kinesins/antagonists & inhibitors , Male , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubules/metabolism , Organ Size , Signal TransductionABSTRACT
BACKGROUND: Triple Negative Breast Cancer (TNBC) is an aggressive and highly heterogeneous subtype of breast cancer associated with poor prognosis. A better understanding of the biology of this complex cancer is needed to develop novel therapeutic strategies for the improvement of patient survival. We have previously demonstrated that Thymoquinone (TQ), the major phenolic compound found in Nigella sativa, induces anti-proliferative and anti-metastatic effects and inhibits in vivo tumor growth in orthotopic TNBC models in mice. Also, we have previously shown that Beclin-1 and LC3 autophagy genes contributes to TNBC cell proliferation, migration and invasion, suggesting that Beclin-1 and LC3 genes provide proto-oncogenic effects in TNBC. However, the role of Beclin-1 and LC3 in mediating TQ-induced anti-tumor effects in TNBC is not known. OBJECTIVE: To investigate the effects of TQ on the major autophagy mediators, Beclin-1 and LC3 expression, as well as autophagic activity in TNBC cells. METHODS: Cell proliferation, colony formation, migration and autophagy activity were evaluated using MTS cell viability, colony formation assay, wound healing and acridine orange staining assays, respectively. Western blotting and RT-PCR assays were used to investigate LC3 and Beclin-1 protein and gene expressions, respectively, in MDA-MB-231 TNBC cells in response to TQ treatments. RESULTS: TQ treatment significantly inhibited cell proliferation, colony formation, migration and autophagic activity of MDA-MB-231 cells and suppressed LC3 and Beclin-1 expressions. Furthermore, TQ treatment led to the inhibition of Integrin-ß1, VEGF, MMP-2 and MMP-9 in TNBC cells. CONCLUSION: TQ inhibits autophagic activity and expression of Beclin-1 and LC3 in TNBC cells and suppresses pathways related to cell migration/invasion and angiogenesis, including Integrin-ß1, VEGF, MMP-2 and MMP- 9, suggesting that TQ may be used to control autophagic activity and oncogenic signaling in TNBC.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Beclin-1/antagonists & inhibitors , Benzoquinones/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Beclin-1/genetics , Beclin-1/metabolism , Benzoquinones/chemical synthesis , Benzoquinones/chemistry , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Molecular Structure , Structure-Activity Relationship , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
Brain tumors are considered as one of the most aggressive and incurable forms of cancer. The majority of the patients with brain tumors have a median survival rate of 12%. Brain tumors are lethal despite the availability of advanced treatment options such as surgical removal, chemotherapy, and radiotherapy. In this study, we have evaluated the anti-cancer effects of pimozide, which is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. Pimozide significantly reduced the proliferation of U-87MG, Daoy, GBM 28, and U-251MG brain cancer cell lines by inducing apoptosis with IC50 (Inhibitory concentration 50) ranging from 12 to 16 µM after 48 h of treatment. Our Western blotting analysis indicated that pimozide suppressed the phosphorylation of STAT3 at Tyr705 and Src at Tyr416, and it inhibited the expression of anti-apoptotic markers c-Myc, Mcl-1, and Bcl-2. Significant autophagy induction was observed with pimozide treatment. LC3B, Beclin-1, and ATG5 up-regulation along with autolysosome formation confirmed the induction of autophagy with pimozide treatment. Inhibiting autophagy using 3-methyladenine or LC3B siRNA significantly blocked the apoptosis-inducing effects of pimozide, suggesting that pimozide mediated its apoptotic effects by inducing autophagy. Oral administration of 25 mg/kg pimozide suppressed the intracranially implanted U-87MG tumor growth by 45% in athymic nude mice. The chronic administration of pimozide showed no general signs of toxicity, and the behavioral activity of the mice remained unchanged. Taken together, these results indicate that pimozide inhibits the growth of brain cancer by autophagy-mediated apoptosis.