67,917 results on '"HeLa Cells"'
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2. Design, Synthesis, and Docking Studies of Thioimidazolyl Diketoacid Derivatives Targeting HIV-1 Integrase
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Nafiseh Karimi, Rouhollah Vahabpour Roudsari, Afshin Zarghi, and Zahra Hajimahdi
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chemistry.chemical_classification ,biology ,Chemistry ,Stereochemistry ,Integrase inhibitor ,HIV Integrase ,biology.organism_classification ,Enzyme assay ,Integrase ,HeLa ,Structure-Activity Relationship ,Enzyme ,Docking (molecular) ,Drug Design ,Drug Discovery ,biology.protein ,Humans ,HIV Integrase Inhibitors ,Cytotoxicity ,IC50 ,HeLa Cells - Abstract
Background: Integrase enzyme is a validated drug target to discover novel structures as anti-HIV-1 agents. Objective: This study aimed at developing a novel series of thioimidazolyl diketoacid derivatives characterizing various substituents at N-1 and 2-thio positions of the central ring as HIV-1integrase inhibitors. Methods: In this study, eighteen novel thioimidazolyl DKA derivatives were synthesized in a fivestep parallel procedure and tested in vitro for the inhibition of both IN ST reaction and the singlecycle HIV-1 replication in HeLa cell culture. Results: The obtained molecules were evaluated using the enzyme assay, displaying promising integrase inhibitory activity with IC50 values ranging from 0.9 to 7.7 mM. The synthesized compounds were also tested for antiviral activity and cytotoxicity using HeLa cells infected by the single-cycle replicable HIV-1 NL4-3. Conclusion: The most potent compound was found to be 18i with EC50 = 19 μM, IC50 = 0.9 μM, and SI = 10.5. Docking studies indicated that the binding mode of the active molecule is well aligned with the known HIV-1integrase inhibitor.
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- 2022
3. A Novel Imidazo[1,2-a]pyridine Compound Reduces Cell Viability and Induces Apoptosis of HeLa Cells by p53/Bax-Mediated Activation of Mitochondrial Pathway
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Bin Xu, Qiuyi Deng, Yang Yu, Xinjie Yang, Yanwen Li, Hua Cao, and Jianwen Mao
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Cancer Research ,Cell Survival ,Pyridines ,Antineoplastic Agents ,Apoptosis ,Flow cytometry ,HeLa ,Western blot ,medicine ,Humans ,MTT assay ,Viability assay ,Cell Proliferation ,bcl-2-Associated X Protein ,Membrane Potential, Mitochondrial ,Pharmacology ,biology ,medicine.diagnostic_test ,Cell growth ,Chemistry ,Cytochrome c ,biology.organism_classification ,Molecular biology ,biology.protein ,Molecular Medicine ,Tumor Suppressor Protein p53 ,HeLa Cells - Abstract
Background: Despite emerging research on new treatment strategies, chemotherapy remains one of the most important therapeutic modalities for cancers. Imidazopyridines are important targets in organic chemistry and, given their numerous applications, they are worthy of attention. Objective: The objective of this study was to design and synthesize a novel series of imidazo[1,2-a]pyridine-derived compounds and investigate their antitumor effects and the underlying mechanisms. Methods: Imidazo[1,2-a]pyridine-derived compounds were synthesized with new strategies and conventional methods. The antitumor activities of the new compounds were evaluated by MTT assay. Flow cytometry and immunofluorescence were performed to examine the effects of the most effective antiproliferative compound on cell apoptosis. Western blot analysis was used to assess the expression of apoptotic proteins. Results: Fifty-two new imidazo[1,2-a]pyridine compounds were designed and successfully synthesized. The compound, 1-(imidazo[1,2-a]pyridin-3-yl)-2-(naphthalen-2-yl)ethane-1,2-dione, named La23, showed high potential for suppressing the viability of HeLa cells (IC50 15.32 μM). La23 inhibited cell proliferation by inducing cell apoptosis, and it reduced the mitochondrial membrane potential of HeLa cells. Moreover, treatment with La23 appeared to increase the expression of apoptotic-related protein P53, Bax, cleaved caspase-3, and cytochrome c at a low concentration range. Conclusion: The novel imidazo[1,2-a]pyridine compound, La23, was synthesized and it suppressed cell growth by inducing cell apoptosis via the p53/Bax mitochondrial apoptotic pathway.
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- 2022
4. Cycloheximide can induce Bax/Bak dependent myeloid cell death independently of multiple BH3-only proteins
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George C.T. Yeoh, David L. Vaux, Paul G Ekert, James E Vince, Joanne H. van Vuuren, Megan Finch-Edmondson, Bernard A. Callus, Katharine J. Goodall, and Ian E Gentle
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0301 basic medicine ,Cell Membranes ,Cultured tumor cells ,lcsh:Medicine ,Apoptosis ,Protein Synthesis ,Biochemistry ,Gene Knockout Techniques ,Mice ,chemistry.chemical_compound ,Animal Cells ,Myeloid Cells ,Cycloheximide ,lcsh:Science ,bcl-2-Associated X Protein ,Uncategorized ,Protein Synthesis Inhibitors ,Multidisciplinary ,Cell Death ,Chemical Synthesis ,Cell biology ,bcl-2 Homologous Antagonist-Killer Protein ,Proto-Oncogene Proteins c-bcl-2 ,Cell Processes ,293T cells ,Cell lines ,Cellular Types ,Cellular Structures and Organelles ,Signal transduction ,biological phenomena, cell phenomena, and immunity ,Biological cultures ,Bcl-2 Homologous Antagonist-Killer Protein ,Research Article ,Proteasome Endopeptidase Complex ,Programmed cell death ,Biosynthetic Techniques ,Protein family ,Bone Marrow Cells ,Biology ,Research and Analysis Methods ,Cell Line ,03 medical and health sciences ,Bcl-2-associated X protein ,Animals ,Humans ,HeLa cells ,Molecular Biology Techniques ,Molecular Biology ,Dose-Response Relationship, Drug ,lcsh:R ,Biology and Life Sciences ,Proteins ,Membrane Proteins ,Cell Biology ,Outer Membrane Proteins ,Cell cultures ,Molecular biology ,030104 developmental biology ,chemistry ,biology.protein ,lcsh:Q ,Apoptosis Regulatory Proteins ,CDK inhibitor ,Cloning - Abstract
Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/- Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.
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- 2023
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5. The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically Unstructured Region That Is Not Involved in Apoptosis Regulation
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Robyn-Lee Burton, Bevan Marshall, Christine J. Hawkins, Stephanie Campbell, Delara Pantaki-Eimany, Michele Barry, Sofia Caria, and Marc Kvansakul
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Models, Molecular ,0301 basic medicine ,Programmed cell death ,Protein Conformation ,Apoptosis ,Vaccinia virus ,Microbiology ,Biochemistry ,Virus ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,X-Ray Diffraction ,Scattering, Small Angle ,Vaccinia ,Humans ,Amino Acid Sequence ,Insect virus ,Molecular Biology ,Caspase ,Uncategorized ,biology ,Cell Biology ,biology.organism_classification ,Molecular biology ,3. Good health ,Autographa californica ,HEK293 Cells ,030104 developmental biology ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,biology.protein ,Signal transduction ,Sequence Alignment ,HeLa Cells - Abstract
Subversion of host cell apoptotic responses is a prominent feature of viral immune evasion strategies to prevent premature clearance of infected cells. Numerous poxviruses encode structural and functional homologs of the Bcl-2 family of proteins, and vaccinia virus harbors antiapoptotic F1L that potently inhibits the mitochondrial apoptotic checkpoint. Recently F1L has been assigned a caspase-9 inhibitory function attributed to an N-terminal α helical region of F1L spanning residues 1–15 (1) preceding the domain-swapped Bcl-2-like domains. Using a reconstituted caspase inhibition assay in yeast we found that unlike AcP35, a well characterized caspase-9 inhibitor from the insect virus Autographa californica multiple nucleopolyhedrovirus, F1L does not prevent caspase-9-mediated yeast cell death. Furthermore, we found that deletion of the F1L N-terminal region does not impede F1L antiapoptotic activity in the context of a viral infection. Solution analysis of the F1L N-terminal regions using small angle x-ray scattering indicates that the region of F1L spanning residues 1–50 located N-terminally from the Bcl-2 fold is an intrinsically unstructured region. We conclude that the N terminus of F1L is not involved in apoptosis inhibition and may act as a regulatory element in other signaling pathways in a manner reminiscent of other unstructured regulatory elements commonly found in mammalian prosurvival Bcl-2 members including Bcl-xL and Mcl-1.
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- 2023
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6. Gold Nanorods are Selective Cytotoxic Agents
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Michael Weinfeld, Mohamed A.M. El Gendy, and Ahmed Abdoon
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Male ,Cancer Research ,Carcinoma, Hepatocellular ,Antineoplastic Agents ,HeLa ,Mice ,Cell Line, Tumor ,Splenocyte ,Animals ,Humans ,Cytotoxic T cell ,Cytotoxicity ,Carcinoma, Renal Cell ,IC50 ,Pharmacology ,Nanotubes ,biology ,Cytotoxins ,Liver Neoplasms ,Cell cycle ,biology.organism_classification ,Kidney Neoplasms ,Cell culture ,Cancer cell ,Cancer research ,Molecular Medicine ,Female ,Gold ,HeLa Cells - Abstract
Background: Gold nanorods (GNRs) are very promising agents with multiple applications in medicine and biology. However, the cytotoxic effects of GNRs have not been fully explored. Objective: Therefore, the main objective of this study was to determine the selective cytotoxic effect of GNRs towards several human tumor cell lines. Methods: To address this issue, three sizes of GNRs (10-nm, 25-nm, and 50-nm) were tested against two human tumor cell lines, namely, human hepatoma HepG2 and human prostate PC3 cancer cells. As GNRs are usually stored in soft tissues inside living bodies, we also tested the effect of GNRs on murine splenocyte viability. To determine if the GNRs displayed selective cytotoxicity towards cancer cells, active GNRs with the size showing the least cytotoxicity to splenocytes were then tested against a panel of 11 human tumor cell lines and two human non-tumor cell lines. Results: Our results showed that the most cytotoxic size of GNRs is 10-nm, followed by the 25-nm GNRs, while the 50-nm GNRs did not show a significant effect. In addition, the 25-nm GNRs were the least cytotoxic to splenocytes when tested for 24 and 48 h. These GNRs showed a selective cytotoxic effect to prostate cancer PC3 cells with median inhibitory concentration (IC50) = 8.3 + 0.37 μM, myeloblastic leukemia HL60 cells (IC50 = 19.7 + 0.89 μM), cervical cancer HeLa cells (IC50 = 24.6 + 0.37 μM), renal adenocarcinoma 786.0 cells (IC50 = 27.34 + 0.6 μM), and hepatoma HepG2 cells (IC50 = 27.79 + 0.03 μM) when compared to the effect on the non-tumor human cells; skin fibroblast BJ cell line (IC50 = 40.13 + 0.7 μM) or epithelial breast MCF10A cells (IC50 = 33.2 + 0.89 μM). High selectivity indices (SIs) were observed in GNRs-treated PC3 and HL60 cells with values ranging from 1.69 to 4.83, whereas moderate SIs were observed in GNRs-treated HeLa, 786.0, and HepG2 cells with values ranging from 1.19 to 1.63. Other cells did not show a similar selective effect, including human laryngeal HEp2 cells, colon HCT116, metastatic renal adenocarcinoma ACHN cells, and human breast cancer cells (MCF7, MDA-MB-231, and MDA-MB-468 cells). The effect of GNRs was confirmed using the colony formation assay and the effect was found to be cell cycle-specific. Finally, it was shown that laser treatment could potentiate the cytotoxic effect of the 25-nm GNRs. Conclusion: GNRs are selective cytotoxic agents and they have the potential to act as candidate anticancer agents.
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- 2022
7. Two novel STAT1 mutations cause Mendelian susceptibility to mycobacterial disease
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Jiarui Wang, Xianqin Zhang, Tingting Zou, Dazhi Zhang, Wenqiang Liu, Zhenxing Liu, Xuejie Peng, Zhengyi Ni, Yang Tan, Meiqi Hou, Mi Zhou, Chao Yuan, and Xiaopei Zhou
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Male ,Mutant ,Biophysics ,Virulence ,Chromosomal translocation ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Protein Domains ,medicine ,Humans ,Genetic Predisposition to Disease ,Amino Acid Sequence ,STAT1 ,Molecular Biology ,Gene ,Cell Nucleus ,Genetics ,Mycobacterium Infections ,Mutation ,Base Sequence ,biology ,Promoter ,DNA ,Cell Biology ,Pedigree ,Protein Transport ,HEK293 Cells ,STAT1 Transcription Factor ,chemistry ,biology.protein ,Female ,Mutant Proteins ,HeLa Cells ,Protein Binding ,Subcellular Fractions - Abstract
Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.
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- 2022
8. Identification of CHMP4C as a new risk gene for inherited dilated cardiomyopathy
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Zilong Qiu, Shengmei Qin, Junbo Ge, Xuejie Li, Bo Yuan, Xianhong Shu, Nianwei Zhou, Shifang Shan, Cuizhen Pan, Xiaolin Wang, Yingying Jiang, and Lu Tang
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Cardiomyopathy, Dilated ,Genetics ,MEDLINE ,medicine ,Humans ,Risk gene ,Identification (biology) ,Dilated cardiomyopathy ,Biology ,Bioinformatics ,medicine.disease ,Molecular Biology ,HeLa Cells - Published
- 2022
9. Novel colchicine derivative CR42-24 demonstrates potent anti-tumor activity in urothelial carcinoma
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Mary M. Hitt, Clayton Bell, Kyle G. Potts, Desmond Pink, Jack A. Tuszynski, and John D. Lewis
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Male ,Cancer Research ,Mice, SCID ,Mice ,chemistry.chemical_compound ,Mice, Inbred NOD ,In vivo ,Cell Line, Tumor ,Animals ,Humans ,Colchicine ,Medicine ,Cytotoxic T cell ,Cisplatin ,Carcinoma, Transitional Cell ,biology ,business.industry ,Xenograft Model Antitumor Assays ,Tubulin Modulators ,Gemcitabine ,In vitro ,Disease Models, Animal ,Tubulin ,Urinary Bladder Neoplasms ,Oncology ,Tolerability ,chemistry ,biology.protein ,Cancer research ,business ,HeLa Cells ,medicine.drug - Abstract
Bladder cancers, and specifically urothelial carcinoma, have few effective treatment options, and tumors typically develop resistance against standard of care chemotherapies leading to significant mortality. The development of alternative therapies with increased selectivity and improved tolerability would significantly impact this patient population. Here, we investigate a novel colchicine derivative, CR42-24, with increased selectivity for the βIII tubulin subtype as a treatment for urothelial carcinoma. βIII tubulin is a promising target due to its low expression in healthy tissues and its clinical association with poor prognosis. This study demonstrated that CR42-24 is selectively cytotoxic to several cancer cell lines at low nanomolar IC50, with high activity in bladder cancer cell lines both in vitro and in vivo. CR42-24 monotherapy in an aggressive urothelial carcinoma xenograft model results in effective control when treated early. We observed significant ablation of large tumors and patient-derived xenografts at low doses with excellent tolerability. CR42-24 was highly synergistic in combination with the standard of care chemotherapies gemcitabine and cisplatin, further increasing its therapeutic potential as a novel treatment for urothelial carcinoma.
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- 2022
10. Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA
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Mohit Jadli, Joni Yadav, Nikita Aggarwal, Arun Chhokar, Veera Mohan Veerapandian, Kulbhushan Thakur, Tejveer Singh, Tanya Tripathi, Alok C. Bharti, and Anjali Bhat
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Cancer Research ,Human papillomavirus ,Uterine Cervical Neoplasms ,Biology ,Exosomes ,Transcriptome ,Cell Line, Tumor ,Next generation sequencing ,medicine ,Genetics ,Biomarkers, Tumor ,Humans ,Reverse transcriptase polymerase chain reaction ,RC254-282 ,Cervical cancer ,Gene Expression Profiling ,RNA ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Oncogene Proteins, Viral ,medicine.disease ,Microvesicles ,Exosome ,Oncology ,Transcript profiling ,Cancer research ,RNA, Viral ,Female ,HeLa Cells - Abstract
Background Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood. Materials and methods The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. Results 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value Conclusion Our data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.
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- 2022
11. Structural mechanism for regulation of Rab7 by site-specific monoubiquitination
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Wookyung Yu, Cheol Yong Choi, Jiseok Baek, Sangho Lee, Su Myung Jung, Jaeeun Jung, Wonjin Yang, Kun Tae, Seok Hee Park, Seungsu Han, and Donghyuk Shin
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biology ,SARS-CoV-2 ,Chemistry ,fungi ,Mutant ,Ubiquitination ,rab7 GTP-Binding Proteins ,Colocalization ,General Medicine ,GTPase ,Endocytosis ,Biochemistry ,Cell biology ,Viral Proteins ,HEK293 Cells ,Ubiquitin ,Structural Biology ,biology.protein ,Humans ,Monoubiquitination ,Rab ,Molecular Biology ,Function (biology) ,HeLa Cells ,Protein Binding - Abstract
Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.
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- 2022
12. NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells
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Richard W. Wong, Kei Makiyama, Akiko Kobayashi, Dominic Chih-Cheng Voon, Masaharu Hazawa, and Keesiang Lim
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NSP9 ,Nuclear Envelope ,Recombinant Fusion Proteins ,Active Transport, Cell Nucleus ,Biophysics ,Viral Nonstructural Proteins ,Cell fate determination ,Endoplasmic Reticulum ,Models, Biological ,Biochemistry ,Article ,Nucleoporin 62 ,Humans ,Nuclear pore ,Nucleoporin ,NUP62 ,Molecular Biology ,Membrane Glycoproteins ,p65 ,Host Microbial Interactions ,biology ,SARS-CoV-2 ,Chemistry ,Endoplasmic reticulum ,Transcription Factor RelA ,COVID-19 ,RNA-Binding Proteins ,Cell Biology ,Cell biology ,Nuclear Pore Complex Proteins ,Cell culture ,Cytoplasm ,Gene Knockdown Techniques ,biology.protein ,Nuclear transport ,HeLa Cells - Abstract
Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.
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- 2022
13. Three human RNA polymerases interact with TFIIH via a common RPB6 subunit
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Hidefumi Suzuki, Yoshifumi Nishimura, Masahiko Okuda, Yuki Yamaguchi, and Tetsufumi Suwa
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AcademicSubjects/SCI00010 ,Protein subunit ,NAR Breakthrough Article ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Genetics ,Humans ,Gene ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Binding Sites ,RNA ,Pleckstrin Homology Domains ,Cell biology ,Molecular Docking Simulation ,Transfer RNA ,Transcription factor II H ,RNA Polymerase II ,Transcription Factor TFIIH ,030217 neurology & neurosurgery ,Nucleotide excision repair ,HeLa Cells ,Protein Binding - Abstract
In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62–RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62–RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.
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- 2022
14. New Findings from Chittaranjan National Cancer Institute in the Area of Experimental Biology Reported (Downregulation of Wnt/ß-catenin Self-renewal Pathway In Cervical Cancer Cells By Polyphenolic Compounds).
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CERVICAL cancer ,CANCER cells ,BIOLOGY ,LOW density lipoprotein receptors ,SOX2 protein ,DOWNREGULATION - Abstract
New Findings from Chittaranjan National Cancer Institute in the Area of Experimental Biology Reported (Downregulation of Wnt/ß-catenin Self-renewal Pathway In Cervical Cancer Cells By Polyphenolic Compounds) West Bengal, India, Asia, Experimental Biology, Life Sciences, Hela Cells, Tumor Cell Line Keywords: West Bengal; India; Asia; Experimental Biology; Life Sciences; Hela Cells; Tumor Cell Line EN West Bengal India Asia Experimental Biology Life Sciences Hela Cells Tumor Cell Line 1077 1077 1 11/06/23 20231110 NES 231110 2023 NOV 10 (NewsRx) -- By a News Reporter-Staff News Editor at Genomics & Genetics Weekly -- Current study results on Life Sciences - Experimental Biology have been published. [Extracted from the article]
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- 2023
15. Production of human translation-competent lysates using dual centrifugation
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Ziegelmüller J, Evangelos D. Karousis, Oliver Mühlemann, and Lukas-Adrian Gurzeler
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Human in vitro translation ,Lysis ,detergent-free cell lysis ,Centrifugation ,In Vitro Techniques ,eIF2-α ,Cell Fractionation ,cap-dependent translation ,Technical Report ,Genes, Reporter ,Gene expression ,540 Chemistry ,Humans ,RNA, Messenger ,Molecular Biology ,Cell-Free System ,Chemistry ,Technical Paper ,Translation (biology) ,Cell Biology ,cell-free translation ,In vitro ,Cell biology ,Cytoplasm ,Protein Biosynthesis ,dual centrifugation ,570 Life sciences ,biology ,HeLa Cells ,Subcellular Fractions - Abstract
Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.
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- 2021
16. The Extensive and Expensive Impacts of HEp-2 [HeLa], Intestine 407 [HeLa], and Other False Cell Lines in Journal Publications
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Amanda Capes-Davis and Christopher Korch
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Cell type ,Biomedical Research ,biology ,Computational biology ,biology.organism_classification ,Biochemistry ,Analytical Chemistry ,Intestines ,HeLa ,Cell culture ,Cell Line, Tumor ,Drug Discovery ,Cervical carcinoma ,Humans ,Molecular Medicine ,Prospective Studies ,Dividing cell ,Literature survey ,HeLa Cells ,Retrospective Studies ,Biotechnology - Abstract
Cell lines are essential models for biomedical research. However, they have a common and important problem that needs to be addressed. Cell lines can be misidentified, meaning that they no longer correspond to the donor from whom the cells were first obtained. This problem may arise due to cross-contamination: the accidental introduction of cells from another culture. The contaminant, which is often a rapidly dividing cell line, will overgrow and replace the original culture. The end result is a false cell line, also known as a misidentified or imposter cell line. False cell lines may come from an entirely different species, tissue, or cell type than the original donor. If undetected, false cell lines produce unreliable and irreproducible results that pollute the biomedical literature and threaten the development of reliable drug discovery and meaningful patient treatments.The goal of this study was to ascertain how widespread this problem is and how it affects the literature, as well as to estimate how much funding has been used to produce pools of scientific literature of questionable value. We focus on HEp-2 [HeLa] and Intestine 407 [HeLa], two false cell lines that are widely used in the scientific literature but were shown to be cross-contaminated in 1967. These two cell lines have been used in 8497 and 1397 published articles and extensively described as laryngeal cancer and normal intestine, respectively, rather than their true identity: the cervical cancer cell line HeLa. Discussed are tools, approaches, and resources that can address this issue-both retrospectively and prospectively.
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- 2021
17. Transmembrane domains of type III-secreted proteins affect bacterial-host interactions in enteropathogenic E. coli
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Dor Braverman, Neta Sal-Man, and Jenia Gershberg
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Microbiology (medical) ,sctb ,Pore complex ,Immunology ,Infectious and parasitic diseases ,RC109-216 ,Biology ,type iii secretion system ,Microbiology ,scte ,Type three secretion system ,Enteropathogenic Escherichia coli ,03 medical and health sciences ,Bacterial Proteins ,Protein Domains ,epec ,Type III Secretion Systems ,Humans ,Secretion ,Enteropathogenic E. coli ,030304 developmental biology ,0303 health sciences ,Host Microbial Interactions ,030306 microbiology ,Effector ,Host (biology) ,Escherichia coli Proteins ,bacterial virulence ,espb ,espd ,transmembrane domain ,Transmembrane domain ,Infectious Diseases ,Secretory protein ,Parasitology ,pore complex ,Bacterial Outer Membrane Proteins ,HeLa Cells ,Research Article ,Research Paper - Abstract
Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.
- Published
- 2021
18. Synthesis, Characterization, Biological Evaluation and Molecular Docking Studies of New Oxoacrylate and Acetamide on HeLa Cancer Cell Lines
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Nevin Çankaya, Serap Yalçın Azarkan, and Mehmetcan İzdal
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In silico ,Protein Data Bank (RCSB PDB) ,Antineoplastic Agents ,HeLa ,Structure-Activity Relationship ,Cell Line, Tumor ,Neoplasms ,Acetamides ,Drug Discovery ,Humans ,Cytotoxicity ,Cell Proliferation ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Cell growth ,Chemistry ,General Medicine ,biology.organism_classification ,In vitro ,Molecular Docking Simulation ,Biochemistry ,Cell culture ,Cancer cell ,Molecular Medicine ,Drug Screening Assays, Antitumor ,HeLa Cells - Abstract
Background: In recent years, discovery and development of new drugs play a critical role in cancer therapy. Objective: In this study, the effect of MPAEA and p-acetamide on cellular toxicity and on silico in HeLa cancer cells have been investigated. Methods: In this study, 2-choloro-N-(4-methoxyphenyl)acetamide (p-acetamide) and 2-(4-methoxyphenylamino)-2- oxoethyl acrylate (MPAEA) have been synthesized and characterized by FTIR, 1H, and 13C-NMR. Cytotoxicity of pacetamide and MPAEA have been investigated by XTT cell proliferation assay on the HeLa cell line. IC50 values of pacetamide and MPAEA have been identified on the HeLa cell line. Further, molecular docking study was carried out by Autodock Vina using BCL-2 (PDB ID: 4MAN), BCL-W (PDB ID: 2Y6W), MCl-1 (PDB ID: 5FDO) AKT (PDB ID: 4GV1) and BRAF (PDB ID: 5VAM) as a possible apoptotic target for anticancer activity. Results: According to the obtained results, MPAEA and p-acetamide were successfully synthesized and characterized. The interactions between ligands and anti-apoptotic proteins were evaluated by molecular docking and their free energy of binding were calculated and used as descriptor. Conclusion: In vitro and in silico the results demonstrated that MPAEA had potent anticancer activity on HeLa cell line.
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- 2021
19. The Combination of Berberine and Methotrexate Enhances Anti-Cancer Effects in Hela Cancer Cell Line: A Morphological Study
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Jafar Ezzati Nazhad Dolatabadi, Mostafa Araj-Khodaei, Dariush Shanehbandi, Maryam Ghaffari, Amir Jafari, Hossein Majidzadeh, and Mohammadali Torbati
- Subjects
biology ,Combination therapy ,hela cells ,Pharmaceutical Science ,Cancer ,nuclear morphology ,biology.organism_classification ,medicine.disease ,methotrexate ,HeLa ,RS1-441 ,chemistry.chemical_compound ,Pharmacy and materia medica ,chemistry ,Apoptosis ,berberine ,Cancer cell ,herbal medicine ,Cancer research ,medicine ,DAPI ,Viability assay ,General Pharmacology, Toxicology and Pharmaceutics ,Cytotoxicity - Abstract
Background: Co-administration of two or several either chemotherapeutic agents or conventional drugs as a combination treatment is the most effective method to increase therapeutic efficiency. Additive or synergistic influence are two mechanisms by which combination therapy causes a rise in optimal cancer therapy compared to a single treatment. Methods: Colorimetric assay was carried out to estimate the cytotoxicity of the combined system, followed by apoptosis assay to calculate the number of apoptotic cells. Both 4′,6-diamidino-2-phenylindole (DAPI) staining and DNA ladder were complemented tests to illuminate morphological changes and DNA fracturing on HeLa cancer cells. Statistical: Through Graph pad Prism 6.0 software. One-way ANOVA was used to determine the significance. A P-value of less than 0.05 was considered to be statistically significant. Results: In this study revealed that the combination of MTX and BER could inhibit the growth of HeLa cancer cells noticeably. Nevertheless, single BER and MTX were not as effective as a combined system to reduce cell viability at the same dose. Regarding the apoptosis induction and change in morphology of cancer cells’ nucleus, co-treatment of BER and MTX was more effective. The result was complemented with flow cytometry, DAPI staining and DNA ladder, which showed that BER+MTX depicted more anti-cancer effects. Conclusion: The combination therapy of HeLa cancer cells with BER and MTX showed high inhibition effect compared to other treated groups.
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- 2021
20. Fis1 phosphorylation by Met promotes mitochondrial fission and hepatocellular carcinoma metastasis
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Xiao Feng Zhu, Zhuo-Yan Zhong, Kaiming Zhang, Xiao-Jun Qian, Yu-Hong Chen, Yan Yu, Shun Li, Xue-Lian Xu, Dong Yang, Li-Huan Zhou, Hai-Liang Zhang, Gong-Kan Feng, Rong Deng, Jun-Dong Li, Xuan Li, Yun-Tian Li, Yun Huang, Xiao-Dan Peng, Zhi-Ling Li, Xiang Huang, and Jia Mai
- Subjects
FIS1 ,Cancer Research ,Carcinoma, Hepatocellular ,QH301-705.5 ,Cell ,Mitochondria, Liver ,Mitochondrion ,Mitochondrial Dynamics ,Receptor tyrosine kinase ,Article ,Metastasis ,Mitochondrial Proteins ,Genetics ,medicine ,Humans ,Phosphorylation ,Biology (General) ,biology ,Chemistry ,Liver Neoplasms ,Membrane Proteins ,Oncogenes ,Proto-Oncogene Proteins c-met ,Cell biology ,medicine.anatomical_structure ,Invadopodia ,biology.protein ,Medicine ,Mitochondrial fission ,Tyrosine kinase ,HeLa Cells - Abstract
Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.
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- 2021
21. RASA4 inhibits the HIFα signaling pathway to suppress proliferation of cervical cancer cells
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Erling Chen, Jinbing Huang, Qiaoqiao Huang, Ji Li, Wensheng Xu, and Junying Chen
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medicine.medical_treatment ,proliferation ,Uterine Cervical Neoplasms ,Bioengineering ,Biology ,rasa4 ,Applied Microbiology and Biotechnology ,Targeted therapy ,HeLa ,Mice ,Cell Line, Tumor ,Survivin ,Basic Helix-Loop-Helix Transcription Factors ,medicine ,hifα ,Animals ,Humans ,Luciferase ,RNA, Messenger ,Cell Proliferation ,Cell growth ,Activator (genetics) ,General Medicine ,Prognosis ,biology.organism_classification ,Hedgehog signaling pathway ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,ras GTPase-Activating Proteins ,Carcinoma, Squamous Cell ,Cancer research ,cervical squamous cell carcinoma ,Female ,Ectopic expression ,TP248.13-248.65 ,Research Article ,Research Paper ,HeLa Cells ,Signal Transduction ,Biotechnology - Abstract
Background RAS p21 protein activator 4 (RASA4) has been recognised as a Ca2+-promoted Ras-MAPK pathway suppressor that inhibits tumour growth. However, the role of RASA4 in cervical squamous cell carcinoma (CESC) remains unclear. Methods The mRNA levels of RASA4 were analysed using the GEO and GEPIA databases. Kaplan-Meier analysis and ROC analyses were conducted to determine the prognostic and diagnostic values for patients from the TCGA-CSCE cohort. The CCK8 and colony assays were performed to assess the impact of RASA4 ectopic expression and gene inactivation on tumour cell proliferation. In vivo experiments were performed. Luciferase reporter assays and LW6 (a HIFα inhibitor) were employed to verify the regulatory relationship between RASA4 and the HIFa signalling pathway. Results The GEPIA and GEO database analysis demonstrated poorly expressed RASA4 in the CESC tissues relative to that in the noncancerous tissues. Based on the TCGA database, poorly expressed RASA4 signified high prognostic and diagnostic values. Ectopically expressed RASA4 weakened the proliferative potential of HeLa cells, whereas RASA4 genetic inactivation produced the opposite impact in the HeLa and C-33A cells. The promoting effect of RASA4 deficiency on tumourigenesis was also recorded in vivo. Subsequently, RASA4 negatively regulated the HIFα-driven luciferase activities and weakened the expression of survivin. Meanwhile, LW6 treatment abrogated the increased proliferation of HeLa cells, as well as the increased expression of survivin by RASA4 depletion. Conclusion Our findings indicated that RASA4 can inhibit the proliferation of cervical cancer cells by inactivating the HIFα signalling pathway, suggesting novel prospects for targeted therapy against CESC.
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- 2021
22. SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy
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Wenqing Ma, Yong Zhao, Yu-wei Gao, Xingyu Li, Wenqi Wang, Hongbin He, Yudong Zhao, Tiecheng Wang, Xiaomei Ma, Xuefeng Wang, Chunqing Xu, Xue Wang, Song Jin, Peili Hou, Hongmei Wang, Zhangping Yu, and Huasong Chang
- Subjects
Proteasome Endopeptidase Complex ,Immunology ,Context (language use) ,Biology ,Virus Replication ,Antiviral Agents ,Article ,Open Reading Frames ,Viral Proteins ,ORF10 ,Interferon ,Mitophagy ,Autophagy ,medicine ,Humans ,Immunology and Allergy ,Gene Silencing ,Adaptor Proteins, Signal Transducing ,Mitochondrial antiviral-signaling protein ,Innate immunity ,Gene knockdown ,Innate immune system ,SARS-CoV-2 ,Ubiquitination ,COVID-19 ,MAVS ,Immunity, Innate ,Mitochondria ,Cell biology ,NIX ,HEK293 Cells ,Infectious Diseases ,Interferon Type I ,Signal transduction ,Infection ,HeLa Cells ,Signal Transduction ,medicine.drug - Abstract
The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe morbidity and mortality in humans. It is urgent to understand the function of viral genes. However, the function of open reading frame 10 (ORF10), which is uniquely expressed by SARS-CoV-2, remains unclear. In this study, we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes. Then, mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway. Furthermore, overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B. Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10. Consistent with our observations, in the context of SARS-CoV-2 infection, ORF10 inhibited MAVS expression and facilitated viral replication. In brief, our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.
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- 2021
23. Research on Radiosensitivity of the Protein Kinase B Signaling Pathway in Cervical Cancer
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Yingping Zhu, Weirong Ma, Leilai Xu, and Zeliang Chen
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MAPK/ERK pathway ,Article Subject ,MAP Kinase Signaling System ,Computer applications to medicine. Medical informatics ,R858-859.7 ,Uterine Cervical Neoplasms ,Radiation Tolerance ,General Biochemistry, Genetics and Molecular Biology ,HeLa ,Cell Line, Tumor ,Humans ,Radiosensitivity ,Telomerase ,Protein kinase B ,General Immunology and Microbiology ,biology ,Cell growth ,Applied Mathematics ,Computational Biology ,General Medicine ,biology.organism_classification ,Gene Expression Regulation, Neoplastic ,Apoptosis ,Gene Knockdown Techniques ,Modeling and Simulation ,Protein kinase B signaling ,Cancer research ,Female ,Signal transduction ,Proto-Oncogene Proteins c-akt ,HeLa Cells ,Signal Transduction ,Transcription Factors ,Research Article - Abstract
The main characteristics of cervical cancer are abnormal and uncontrolled cell proliferation, and it regulates cell growth, differentiation, and cell death through genetic and epigenetic changes. This paper mainly discusses the radiosensitivity of the cervical cancer protein kinase B signaling pathway and discusses the specific mechanisms that affect the occurrence and development of cervical cancer. In addition, this paper studies the effect of transient transfection knocking down the expression of TRIP4 in cervical cancer cells on the expression of key proteins in related signaling pathways and explores the mechanism of its specific effects and finds the mechanism of TRIP4’s effect on cervical cancer radiosensitivity. The findings of this study show for the first time that knocking down TRIP4 inhibits cell viability by inhibiting the P13K/AKT and MAPK/ERK pathways, and this corresponds to the first part of the experimental results, which show that knocking down TRIP4 inhibits colony formation and increases apoptosis in HeLa and SiHa cells. Moreover, simultaneous inhibition of TRIP4 and hTERT proteins can increase the radiosensitivity of cervical cancer cells. These findings indicate that the inhibition of TRIP4 may be a new type of treatment that selectively targets the P13K/AKT and MAPK/ERK pathways and hTERT pathways in cervical cancer cells and provides a therapeutic option for the treatment of cervical cancer.
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- 2021
24. Inhibitor of growth protein 3 epigenetically silences endogenous retroviral elements and prevents innate immune activation
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Stephen M. Beverley, Natalia S. Akopyants, Siyuan Ding, Yaw Shin Ooi, Jan E. Carette, Jonathan Diep, Gaopeng Hou, Yanhua Song, and Harry B. Greenberg
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Homeodomain Proteins ,Innate immune system ,AcademicSubjects/SCI00010 ,Somatic cell ,Tumor Suppressor Proteins ,Gene regulation, Chromatin and Epigenetics ,Endogenous Retroviruses ,Endogenous retrovirus ,Promoter ,Endogeny ,Biology ,Immunity, Innate ,Virus ,Cell biology ,Histone Code ,Narese/2 ,Narese/1 ,Genetics ,Humans ,Gene silencing ,Gene Silencing ,CRISPR-Cas Systems ,HT29 Cells ,Function (biology) ,HeLa Cells - Abstract
Endogenous retroviruses (ERVs) are subject to transcriptional repression in adult tissues, in part to prevent autoimmune responses. However, little is known about the epigenetic silencing of ERV expression. Here, we describe a new role for inhibitor of growth family member 3 (ING3), to add to an emerging group of ERV transcriptional regulators. Our results show that ING3 binds to several ERV promoters (for instance MER21C) and establishes an EZH2-mediated H3K27 trimethylation modification. Loss of ING3 leads to decreases of H3K27 trimethylation enrichment at ERVs, induction of MDA5-MAVS-interferon signaling, and functional inhibition of several virus infections. These data demonstrate an important new function of ING3 in ERV silencing and contributing to innate immune regulation in somatic cells.
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- 2021
25. Inhibition of the futalosine pathway for menaquinone biosynthesis suppresses Chlamydia trachomatis infection
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David Needham, Dewey G. McCafferty, Taylor C. Outlaw, Brianne M. Dudiak, and Tri M. Nguyen
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Docosahexaenoic Acids ,medicine.drug_class ,Antibiotics ,Biophysics ,Chlamydia trachomatis ,Immunofluorescence ,medicine.disease_cause ,Biochemistry ,Article ,HeLa ,Automation ,Structural Biology ,Genetics ,medicine ,Humans ,Molecular Biology ,Inclusion Bodies ,chemistry.chemical_classification ,medicine.diagnostic_test ,biology ,Nucleosides ,Vitamin K 2 ,Cell Biology ,Chlamydia Infections ,biology.organism_classification ,Anti-Bacterial Agents ,Biosynthetic Pathways ,Futalosine ,Enzyme ,chemistry ,Docosahexaenoic acid ,Nanoparticles ,Bacteria ,HeLa Cells - Abstract
Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow-spectrum antibiotic targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through liquid chromatography-tandem mass spectrometry. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with the futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.
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- 2021
26. Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs)
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Angela Gong, Elvira An, Ifunanya Okeke, Saul Jaime-Figueroa, Zhenyi Hu, Xuanmeng Luo, Craig M. Crews, Po-Han Chen, and Sijin Zheng
- Subjects
Phosphatase ,Hyperphosphorylation ,Biochemistry ,Dephosphorylation ,Structure-Activity Relationship ,Catalytic Domain ,Humans ,Protein phosphorylation ,Phosphorylation ,biology ,Chimera ,Chemistry ,Kinase ,Forkhead Box Protein O3 ,Retinoblastoma protein ,RNA-Binding Proteins ,General Medicine ,Protein phosphatase 2 ,Phosphoproteins ,Cell biology ,Enzyme Activation ,biology.protein ,Molecular Medicine ,Protein Tyrosine Phosphatases ,Apoptosis Regulatory Proteins ,Holoenzymes ,HeLa Cells - Abstract
Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.
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- 2021
27. A Novel <scp> TFG </scp> Mutation in a Korean Family with <scp>α‐Synucleinopathy</scp> and Amyotrophic Lateral Sclerosis
- Author
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Tae-Beom Ahn, Wonjae Lee, Hyo Sun Kim, Dallah Yoo, Jungho Kim, Seung-Jae Lee, Chaewon Shin, Jae-Jun Ban, Gye Sun Jeon, and Jung-Joon Sung
- Subjects
Proband ,Mutation ,Synucleinopathies ,Parkinsonism ,Endoplasmic reticulum ,Amyotrophic Lateral Sclerosis ,Neurodegeneration ,Proteins ,Biology ,medicine.disease ,medicine.disease_cause ,Multisystem proteinopathy ,Atrophy ,Neurology ,Republic of Korea ,Cancer research ,medicine ,Humans ,Neurology (clinical) ,Amyotrophic lateral sclerosis ,HeLa Cells - Abstract
Background Tropomyosin-receptor kinase fused gene (TFG) functions as a regulator of intracellular protein packaging and trafficking at the endoplasmic reticulum exit sites. TFG has recently been proposed as a cause of multisystem proteinopathy. Objectives Here, we describe a Korean family presenting with Parkinson's disease or amyotrophic lateral sclerosis caused by a novel variant of TFG (c.1148 G > A, p.Arg383His). Methods We collected clinical, genetic, dopamine transporter imaging, nerve conduction, and electromyography data from the seven subjects. To verify the pathogenicity of the R383H variant, we studied cell viability and the abnormal aggregation of α-synuclein and TAR DNA-binding protein 43 (TDP-43) in HeLa cells expressing R383H-TFG. Results The clinical phenotypes of the R383H-TFG mutation varied; of the five family members, one had Parkinson's disease, three had subclinical parkinsonism, and one (the proband) had amyotrophic lateral sclerosis. The individual with multiple system atrophy was the proband's paternal cousin, but the TFG genotype was not confirmed due to unavailability of samples. Our in vitro studies showed that R383H-TFG overexpression impaired cell viability. In cells co-expressing R383H-TFG and α-synuclein, insoluble α-synuclein aggregates increased in concentration and were secreted from the cells and co-localized with R383H-TFG. The levels of cytoplasmic insoluble aggregates of TDP-43 increased in HeLa cells expressing R383H-TFG and co-localized with R383H-TFG. Conclusions Clinical and in vitro studies have supported the pathogenic role of the novel TFG mutation in α-synucleinopathy and TDP-43 proteinopathy. These findings expand the phenotypic spectrum of TFG and suggest a pivotal role of endoplasmic reticulum dysfunction during neurodegeneration. © 2021 International Parkinson and Movement Disorder Society.
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- 2021
28. Transcriptomic analysis reveals that coxsackievirus B3 Woodruff and GD strains use similar key genes to induce FoxO signaling pathway activation in HeLa cells
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Mi Liu, Jun Han, and Qian Yang
- Subjects
GABARAP ,Coxsackievirus Infections ,PIM1 ,General Medicine ,Cell cycle ,Biology ,Virology ,Enterovirus B, Human ,Cell biology ,ATG12 ,Transcriptome ,Myocarditis ,Humans ,Signal transduction ,KEGG ,Gene ,HeLa Cells ,Signal Transduction - Abstract
Coxsackievirus B3 (CVB3) is a major cause of viral myocarditis in humans. Although there have been studies on CVB3 infection and pathogenesis, the precise disease mechanism is still not clear. In this study, we used RNA-seq technology to compare the transcriptomic profile of virus-infected HeLa cells to that of uninfected cells to identify key genes involved in host-virus interaction. For this, two CVB3 strains, CVB3 Woodruff, an experimental strain, and GD16-69/GD/CHN/2016, a clinical strain, were selected to examine the common mechanisms underlying their infection. Transcriptomic profiles revealed increased expression of the cell cycle genes CCNG2, GADD45B, PIM1, RBM15, KLF10, and RIOK3 and decreased expression of CYBA. The autophagy-related genes ATG12 and YOD1 were found to be upregulated, while the expression of SOD2 and XPO1 increased slightly in infected cells, and only a minor change was observed in GABARAP expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the FoxO signaling pathway to be enriched and showed a close interaction with differentially expressed genes (DEGs) in the protein-protein interaction network. DEGs associated with related pathways such as cell cycle, autophagy, and oxidative stress resistance were also confirmed by qRT-PCR. In summary, the FoxO signaling pathway was activated during infection with both CVB3 strains and was found to have a regulatory role in downstream pathways such as cell cycle, autophagy, oxidative stress resistance, and the antiviral immune response.
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- 2021
29. MiR-106b-5p Promotes Malignant Behaviors of Cervical Squamous Cell Carcinoma Cells by Targeting TIMP2
- Author
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Xuejun Chen and Huier Sun
- Subjects
Tissue Inhibitor of Metalloproteinase-2 ,Cervical Squamous Cell Carcinoma ,medicine.diagnostic_test ,Cell growth ,Uterine Cervical Neoplasms ,Obstetrics and Gynecology ,Apoptosis ,Matrix metalloproteinase ,Biology ,Phenotype ,Cell Line ,Gene Expression Regulation, Neoplastic ,MicroRNAs ,Rna immunoprecipitation ,Mrna level ,Western blot ,Carcinoma, Squamous Cell ,medicine ,Cancer research ,Humans ,Female ,Mir 106b ,Cell Proliferation ,HeLa Cells - Abstract
The objective of this study was to investigate modulatory mechanism of miR-106b-5p and tissue inhibitor of metalloproteinases 2 (TIMP2) on cervical squamous cell carcinoma cells. Differentially expressed genes in CSCC were analyzed via bioinformatics analysis. The targeting impact of miR-106b-5p on TIMP2 was validated through dual-luciferase assay and RNA immunoprecipitation assay. MiR-106b-5p level and TIMP2 mRNA level were assessed via qRT-PCR. TIMP2 protein level was measured via western blot. Malignant behaviors of CSCC cells were evaluated by functional experiments. The EMT and apoptosis-related proteins were determined via western blot. MiR-106b-5p was noticeably elevated in CSCC cells. Its downstream target was TIMP2. MiR-106b-5p and TIMP2 levels were inversely correlated. MiR-106b-5p overexpression fostered malignant phenotypes of CSCC cells, and vice versus. TIMP2 overexpression weakened the promotive impact of forced expression of miR-106b-5p on CSCC cell growth. EMT was facilitated by forced expression of miR-106b-5p. MiR-106b-5p regulates the progression of CSCC cells via targeting TIMP2, which may provide novel value for development of therapeutic targets for CSCC.
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- 2021
30. Potential in vitro therapeutic effects of targeting SP/NK1R system in cervical cancer
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Mahtab Mozafari, Seyed Isaac Hashemy, Reza Assaran Darban, and Safieh Ebrahimi
- Subjects
Gene Expression ,Uterine Cervical Neoplasms ,Apoptosis ,Substance P ,HeLa ,Neurokinin-1 Receptor Antagonists ,Downregulation and upregulation ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,MTT assay ,Viability assay ,Molecular Biology ,Aprepitant ,Cell Proliferation ,biology ,Chemistry ,Cell growth ,General Medicine ,Receptors, Neurokinin-1 ,Cell cycle ,biology.organism_classification ,Gene Expression Regulation, Neoplastic ,Matrix Metalloproteinase 9 ,Cancer research ,Matrix Metalloproteinase 2 ,Female ,HeLa Cells ,Signal Transduction ,medicine.drug - Abstract
Cervical cancer, an aggressive gynecological cancer, seriously threatens women’s health worldwide. It is recently reported that neuropeptide substance P (SP) regulates many tumor-associated processes through neurokinin-1 receptor (NK1R). Therefore, we used cervical cancer cell line (HeLa) to investigate the functional relevance of the SP/NK1R system in cervical cancer pathogenesis. Cellular proliferation and cytotoxicity were analyzed by colorimetric MTT assay. Quantitative real-time PCR (qRT-PCR) was used to measure mRNA expression levels of desired genes. Cell cycle distribution and apoptosis were evaluated by flow cytometry. A wound-healing assay was employed to assess migration ability. We found that the truncated isoform of NK1R(NK1R-Tr) is the dominantly expressed form of the receptor in Hela cells. We also indicated that that SP increased HeLa cell proliferation while treatment with NK1R antagonist, aprepitant, inhibited HeLa cell viability in a dose and time-dependent manner. SP also alters the levels of cell cycle regulators (up-regulation of cyclin B1 along with downregulation of p21) and apoptosis-related genes (up-regulation of Bcl-2 along with downregulation of Bax) while aprepitant reversed these effects. Aprepitant also induced arrest within the G2 phase of the cell cycle and subsequent apoptosis. Furthermore, SP promoted the migrative phenotype of HeLa cells and increased MMP-2 and MMP-9 expression while aprepitant exposure significantly reversed these effects. Collectively, our results indicate the importance of the SP / NK1R system in promoting both proliferative and migrative phenotypes of cervical cancer cells and suggest that aprepitant may be developed as a novel treatment for combating cervical cancer.
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- 2021
31. Nsp1 of SARS-CoV-2 stimulates host translation termination
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Aleksandra Arnautova, Elena Alkalaeva, Konstantin Evmenov, Nikita Biziaev, Vera A. Matrosova, Alexey Shuvalov, Tatiana Egorova, Elizaveta Sokolova, Ekaterina Shuvalova, and Nikolay Pustogarov
- Subjects
Protein Conformation ,viruses ,Protein subunit ,Viral Nonstructural Proteins ,Ribosome ,GTP Phosphohydrolases ,Eukaryotic translation ,Protein Domains ,Animals ,Humans ,Eukaryotic Small Ribosomal Subunit ,RNA, Messenger ,Molecular Biology ,Cell-Free System ,biology ,SARS-CoV-2 ,virus diseases ,Translation (biology) ,Cell Biology ,Peptide Chain Termination, Translational ,Stop codon ,Cell biology ,ABCE1 ,Protein Biosynthesis ,Mutation ,Codon, Terminator ,biology.protein ,Rabbits ,Peptides ,Eukaryotic Ribosome ,Ribosomes ,Research Paper ,HeLa Cells ,Peptide Termination Factors ,Protein Binding - Abstract
Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.
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- 2021
32. Mitoquinone Inactivates Mitochondrial Chaperone TRAP1 by Blocking the Client Binding Site
- Author
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Changwook Lee, Byoung Heon Kang, Hakbong Lee, Ki Bum Hong, Sujae Yang, Byung-Gyu Kim, Soosung Kang, Sung Hu, Ji-Hoon Lee, Kyungjae Myung, Soyeon Kim, Nam Gu Yoon, and Darong Kim
- Subjects
Ubiquinone ,Mice, Nude ,Antineoplastic Agents ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Organophosphorus Compounds ,Colloid and Surface Chemistry ,Neoplasms ,Heat shock protein ,Animals ,Humans ,HSP90 Heat-Shock Proteins ,Binding site ,MitoQ ,Binding Sites ,biology ,Chemistry ,General Chemistry ,Xenograft Model Antitumor Assays ,Hsp90 ,In vitro ,Cell biology ,Chaperone (protein) ,Cancer cell ,biology.protein ,Tumor necrosis factor alpha ,HeLa Cells - Abstract
Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitro and in vivo than previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.
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- 2021
33. The human telomeric proteome during telomere replication
- Author
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Alexandra Vancevska, Joachim Lingner, Chih-Yi Gabriela Lin, Anna Christina Naeger, Thomas Lunardi, and Gérald Lossaint
- Subjects
DNA Replication ,Telomerase ,strand synthesis ,AcademicSubjects/SCI00010 ,Telomere-Binding Proteins ,Biology ,Genome ,Shelterin Complex ,Histones ,dna-replication ,repeat-containing rna ,03 medical and health sciences ,0302 clinical medicine ,Histone H1 ,Replication (statistics) ,Genetics ,Humans ,trf1 ,Telomere Shortening ,030304 developmental biology ,chromatin compaction ,Telomere-binding protein ,0303 health sciences ,blm helicase ,Gene regulation, Chromatin and Epigenetics ,DNA replication ,dynamics ,Telomere ,proteins ,Cell biology ,HEK293 Cells ,histone chaperone ,Proteome ,Replisome ,complex ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.
- Published
- 2021
34. Erythrokeratodermia variabilis et progressiva due to a novel mutation in GJB4
- Author
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Peng Xu, Yuling Shi, Yangfeng Ding, Jiajing Lu, Jun Gu, and Xilin Zhang
- Subjects
Heterozygote ,Epidermis (botany) ,Genetic heterogeneity ,Mutant ,Mutation, Missense ,Genodermatosis ,Connexin ,Dermatology ,Biology ,medicine.disease ,Biochemistry ,Penetrance ,Molecular biology ,Connexins ,medicine ,Humans ,Missense mutation ,Erythrokeratodermia Variabilis ,Molecular Biology ,Gene ,HeLa Cells - Abstract
Erythrokeratodermia variabilis et progressiva (EKVP) is a rare genodermatosis of clinical and genetic heterogeneity, characterized by the manifestations of localized or disseminated persistent hyperkeratotic plagues and stationary to migratory transient erythematous patches. The majority of EKVP cases display an autosomal dominant mode of inheritance with incomplete penetrance, although recessive transmission has also been described. Mutations associated with EKVP have been primarily detected in connexin (Cx) genes. We herein reported a Chinese sporadic case of late-onset EKVP with a novel heterozygous missense mutation c.109G>A (p.V37M) in GJB4 (Cx30.3) gene, which resulted in a significant reduction of GJB4 expression in the epidermis of the patient. In accordance, while wild-type GJB4 localized at the cell membrane of HeLa cells forming intercellular junctions and intracellular puncta, V37M mutant variant was diffusely expressed within HeLa cells at a considerably lower level. Our findings reveal an essential role of GJB4 in the pathogenesis of EKVP and provides insights into the therapeutic potential of the disease.
- Published
- 2021
35. Inhibition of Autophagy by a Small Molecule through Covalent Modification of the LC3 Protein
- Author
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Zhifeng Chen, Bing Zhou, Cheng Luo, Junchi Hu, Chinatsu Otomo, Xie Yuli, Yongjun Dang, Liyan Yue, Yuanyuan Zhang, Lianchun Li, Naixia Zhang, Wei Wan, Junyan Lu, Hualiang Jiang, Takanori Otomo, Yi Wen, Quanfu Li, Lin Tingting, Hongru Tao, Zhiyi Yao, Hong Ding, Pan Xu, Mingrui Zhu, Bidong Zhang, Minjia Tan, Kaixian Chen, and Shijie Fan
- Subjects
Models, Molecular ,Molecular Structure ,biology ,Chemistry ,Autophagy ,Lipid-anchored protein ,General Medicine ,General Chemistry ,biology.organism_classification ,Small molecule ,Article ,Catalysis ,Cell biology ,Small Molecule Libraries ,HeLa ,Covalent bond ,Acetylation ,Proteome ,Humans ,Receptor ,Microtubule-Associated Proteins ,HeLa Cells - Abstract
The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins, including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering with the interface. Through screening covalent compounds, we discovered a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.
- Published
- 2021
36. Spectral Characterization of Purpurin Dye and Its Application in pH Sensing, Cell Imaging and Apoptosis Detection
- Author
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D. Suresh, Susobhan Mahanty, and Krishnan Rathinasamy
- Subjects
Absorption (pharmacology) ,Magnetic Resonance Spectroscopy ,Sociology and Political Science ,Intracellular pH ,Clinical Biochemistry ,Anthraquinones ,Apoptosis ,Protonation ,Biosensing Techniques ,Biochemistry ,HeLa ,Fluorescence microscope ,Animals ,Humans ,Coloring Agents ,Spectroscopy ,biology ,Chemistry ,Hydrogen-Ion Concentration ,Plants ,biology.organism_classification ,Molecular Imaging ,Clinical Psychology ,Microscopy, Fluorescence ,Cell culture ,Proton NMR ,Hypsochromic shift ,Law ,Social Sciences (miscellaneous) ,HeLa Cells ,Nuclear chemistry - Abstract
Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a natural red dye obtained from the red madder plant that is widely used in food and dyeing industries. The present study investigated the characteristics of purpurin and its application as a pH-sensitive probe to detect the pH of solutions and intracellular pH of mammalian and bacterial cells. Purpurin exhibited high pH-sensitive behavior, low analytes interference, high stability with pKa of 4.6 and visible colorimetric change. 1H NMR and FTIR studies indicated protonation of phenolic hydroxyl group under acidic condition with hypsochromic shift in the absorption and fluorescence spectra relative to that of basic condition. Cell culture studies using HeLa cells revealed that purpurin is well tolerated by the cells and the fluorescent imaging result indicated excellent cell permeability with possible use of the dye to detect the pH fluctuations in living cells under various physiological conditions such as apoptosis. Microbiological studies indicated that the dye could be used for visualization of bacteria under acidic condition.
- Published
- 2021
37. Hypoxia-responsive pullulan-based nanoparticles as erlotinib carriers
- Author
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Yoshihiro Ito, Mohammed A. Abosheasha, Hriday Bera, and Motoki Ueda
- Subjects
media_common.quotation_subject ,Antineoplastic Agents ,Endocytosis ,Biochemistry ,Clathrin ,HeLa ,Erlotinib Hydrochloride ,chemistry.chemical_compound ,Structural Biology ,Humans ,Internalization ,Glucans ,Molecular Biology ,media_common ,biology ,Chemistry ,Pinocytosis ,Pullulan ,General Medicine ,biology.organism_classification ,Drug Liberation ,Apoptosis ,Drug delivery ,biology.protein ,Biophysics ,Nanoparticles ,Tumor Hypoxia ,HeLa Cells - Abstract
A hypoxia-responsive pullulan-based co-polymer was developed to assess its efficacy to deliver erlotinib (ERL) to the cervical cancer cells. Upon exposure to hypoxic condition, the synthesized and structurally characterized co-polymer i.e. succinyl pullulan-g-6-(2-nitroimidazole) hexylamine (Pull-SA-HA-NI) exhibited a hypochromic shift in the UV spectra and alteration in its self-assembled structures as compared to the control co-polymer, succinyl pullulan-g-hexylamine (Pull-SA-HA). Its corresponding ERL-loaded nanoparticles (NPs) displayed an attenuated crystallinity of pure ERL with excellent drug-trapping capacity (DEE, 94.23 ± 1.36%) and acceptable zeta potential (+39.21 ± 1.09 mV) and diameter (84.10 ± 2.10 nm) values. These also evidenced a faster drug release profile under hypoxic condition relative to the normoxic condition. The cellular internalization of the NPs was mediated through the energy-dependent endocytic process, which could utilize its multiple pathways (i.e., macropinocytosis, clathrin- and caveolae-mediated endocytosis). The ERL-loaded NPs suppressed HeLa cell proliferation and induced apoptosis more efficiently than the pristine drug.
- Published
- 2021
38. Carvacrol Exhibits Chemopreventive Potential against Cervical Cancer Cells via Caspase-Dependent Apoptosis and Abrogation of Cell Cycle Progression
- Author
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Irfan A. Ansari and Afza Ahmad
- Subjects
Cancer Research ,Cell Survival ,Uterine Cervical Neoplasms ,Antineoplastic Agents ,Apoptosis ,medicine.disease_cause ,HeLa ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Tumor Cells, Cultured ,medicine ,Humans ,MTT assay ,Carvacrol ,Viability assay ,DAPI ,Cell Proliferation ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Dose-Response Relationship, Drug ,biology ,Chemistry ,Cell Cycle ,Cell cycle ,biology.organism_classification ,Caspase Inhibitors ,030220 oncology & carcinogenesis ,Cancer research ,Cymenes ,Molecular Medicine ,Female ,Drug Screening Assays, Antitumor ,Reactive Oxygen Species ,Carcinogenesis ,HeLa Cells - Abstract
Background: The carcinogenesis of the uterine cervix is predominantly initiated with the consistent infection of the Human Papilloma Virus (HPV). Owing to the side effects of standard chemotherapeutics in the treatment of recurrent and metastatic cervical cancer, there is a need for a better and effective treatment modality. In this lieu of concern, natural compounds have proven their worthwhile potential against the treatment of various carcinomas. Carvacrol is a phenolic monoterpenoid and several reports have suggested its different biological properties including antioxidant, anti-inflammatory and anticancer activity. Objective: The objective of our present study was to investigate the effect of carvacrol on HPV18+ HeLa cervical cancer cells. Methods: HeLa cervical cancer cells were cultured and subsequently treated with various doses of carvacrol. Cell viability was assessed via MTT assay. DAPI and Hoechst3342 staining were used to qualitatively analyzed the induced apoptosis. Reactive Oxygen Species (ROS) was estimated by DCFDA staining protocol and quantitatively estimated by flow cytometry. The cell cycle distribution and apoptosis (FITC-Annexin V assay) were analyzed by flow cytometry. Results: The results of the present study have established that carvacrol strongly suppresses the proliferation of cervical cancer cells via caspase-dependent apoptosis and abrogation of cell cycle progression. Furthermore, our preliminary study also demonstrated that carvacrol exhibits a synergistic effect with chemotherapeutic drugs (5-FU and carboplatin). These initial findings implicated that natural compounds could reduce the toxic effects of chemotherapeutic drugs. Conclusion: Therefore, this investigation affirms the anti-cancer potential of carvacrol against cervical cancer cells, which could be an appendage in the prevention and treatment of cervical cancer.
- Published
- 2021
39. Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA
- Author
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Jennifer Seematti Sundar, Yasmeen Khan, Souvik Maiti, Arpita Ghosh, Mary Krishna Ekka, Debojyoti Chakraborty, Asgar Hussain Ansari, Praveen Singh, and Satyaprakash Pandey
- Subjects
Gene knockdown ,MALAT1 ,Nucleophosmin ,AcademicSubjects/SCI00010 ,Alternative splicing ,RNA-Binding Proteins ,Biology ,G-quadruplex ,Phosphoproteins ,Cell biology ,G-Quadruplexes ,Alternative Splicing ,RNA splicing ,Genetics ,RNA and RNA-protein complexes ,Humans ,RNA, Long Noncoding ,Nucleolin ,Gene ,HeLa Cells - Abstract
MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3′ region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3′ region of MALAT1 orchestrating AS.
- Published
- 2021
40. Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection
- Author
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Yujin Shi, Wensheng Wei, Tao Jiao, Tian Zheng, Ruiyi Ma, Lili Ren, Qi Jiang, Zichun Xiang, Zhixun Dou, Jian Rao, Zhuo Zhou, Xinyi Zhang, Zhengfan Jiang, Jianwei Wang, Wenjing Wang, Xia Xiao, Tao Deng, Xiaojing Dong, and Xiaobo Lei
- Subjects
Cancer Research ,Cytoplasm ,QH301-705.5 ,viruses ,Mice, Transgenic ,Biology ,Article ,Mice ,Immune system ,Interferon ,Gene expression ,Genetics ,medicine ,Animals ,Humans ,Biology (General) ,Innate immunity ,Innate immune system ,Cell fusion ,SARS-CoV-2 ,HEK 293 cells ,COVID-19 ,Nucleotidyltransferases ,Chromatin ,Immunity, Innate ,Cell biology ,Disease Models, Animal ,HEK293 Cells ,Viral replication ,Infectious diseases ,Medicine ,medicine.drug ,HeLa Cells - Abstract
The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain to be determined. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells’ self-nucleic acids can be employed as a “danger signal” to alarm the immune system.
- Published
- 2021
41. The anti-neoplastic activities of aloperine in HeLa cervical cancer cells are associated with inhibition of the IL-6-JAK1-STAT3 feedback loop
- Author
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Wen-Wen Fan, Kun Xu, Fang-Yu Cai, Hui Zhang, Yao-Dong Chen, Yu-Ze Mao, Xiaofang Liu, Feng-Qi Jiang, Yongsheng Yang, and Wu Chen
- Subjects
STAT3 Transcription Factor ,Quinolizidines ,Mice, Nude ,Uterine Cervical Neoplasms ,Apoptosis ,Feedback ,Flow cytometry ,HeLa ,Mice ,Western blot ,Cell Movement ,In vivo ,Cell Line, Tumor ,Drug Discovery ,medicine ,Animals ,Humans ,MTT assay ,Epithelial–mesenchymal transition ,STAT3 ,Cell Proliferation ,medicine.diagnostic_test ,biology ,Interleukin-6 ,Chemistry ,Janus Kinase 1 ,General Medicine ,biology.organism_classification ,Complementary and alternative medicine ,Cancer research ,biology.protein ,Female ,HeLa Cells ,Signal Transduction - Abstract
Cervical cancer (CC) is recognized as the most common neoplasm in the female reproductive system worldwide. The lack of chemotherapeutic agents with outstanding effectiveness and safety severely compromises the anti-cipated prognosis of patients. Aloperine (ALO) is a natural quinolizidine alkaloid with marked anti-cancer effects on multiple malignancies as well as favorable activity in relieving inflammation, allergies and infection. However, its therapeutic efficacy and underlying mechanism in CC are still unclear. In the current study, MTT assay was employed to evaluate the viability of HeLa cells exposed to ALO to preliminarily estimate the effectiveness of ALO in CC. Then, the effects of ALO on the proliferation and apoptosis of HeLa cells were further investigated by plate colony formation and flow cytometry, respectively, while the migration and invasion of ALO-treated HeLa cells were evaluated using Transwell assay. Moreover, nude mice were subcutaneously inoculated with HeLa cells to demonstrate the anti-CC properties of ALO in vivo. The molecular mechanisms underlying these effects of ALO were evaluated by Western blot and immunohistochemical analysis. This study experimentally demonstrated that ALO inhibited the proliferation of HeLa cells via G2 phase cell cycle arrest. Simultaneously, ALO promoted an increase in the percentage of apoptotic HeLa cells by increasing the Bax/Bcl-2 ratio. Additionally, the migration and invasion of HeLa cells were attenuated by ALO treatment, which was considered to result from inhibition of epithelial-to-mesenchymal transition. For molecular mechanisms, the expression and activation of the IL-6-JAK1-STAT3 feedback loop were markedly suppressed by ALO treatment. This study indicated that ALO markedly suppresses the proliferation, migration and invasion and enhances the apoptosis of HeLa cells. In addition, these prominent anti-CC properties of ALO are associated with repression of the IL-6-JAK1-STAT3 feedback loop.
- Published
- 2021
42. Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling
- Author
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Hongyan Wang, Robert G. Roeder, Zhong Chen, Yue Zhao, William Hankey, Alexandre Rosa Campos, Jiaoti Huang, Qianben Wang, Jeff Groth, and Furong Huang
- Subjects
Male ,Transcription, Genetic ,Molecular biology ,Science ,General Physics and Astronomy ,RNA polymerase II ,Biochemistry ,Article ,General Biochemistry, Genetics and Molecular Biology ,Transcription (biology) ,Cell Line, Tumor ,Neoplasms ,Gene expression ,Humans ,Gene silencing ,Prostate tumors ,Cancer ,Cellular transcription ,Multidisciplinary ,biology ,Chemistry ,Paf1 complex ,Nuclear Proteins ,General Chemistry ,In vitro ,Cell biology ,biology.protein ,RNA Polymerase II ,HeLa Cells ,Transcription Factors - Abstract
RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states., RNA Polymerase II (Pol II) recycling can influence transcription efficiency. Here the authors describe an approach aimed at facilitating the identification of factors involved in Pol II recycling and identify PAF1 complex components as mediators of recycling.
- Published
- 2021
43. Genetic fusions favor tumorigenesis through degron loss in oncogenes
- Author
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Collin Tokheim, Jonathan D. Lee, Wenyi Wei, Jing Liu, X. Shirley Liu, Wenjian Gan, Pier Paolo Pandolfi, and Brian J. North
- Subjects
Male ,Oncogene Proteins, Fusion ,Ubiquitylation ,Carcinogenesis ,Science ,Amino Acid Motifs ,Transplantation, Heterologous ,General Physics and Astronomy ,Mice, Nude ,Computational biology ,Protein degradation ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Article ,Transcription (biology) ,Cell Line, Tumor ,Neoplasms ,medicine ,Cancer genomics ,Animals ,Humans ,Gene ,Cancer genetics ,Cells, Cultured ,Mice, Knockout ,Multidisciplinary ,Oncogene ,Models, Genetic ,Mechanism (biology) ,Computational Biology ,General Chemistry ,Oncogenes ,HCT116 Cells ,Fusion protein ,Gene Expression Regulation, Neoplastic ,HEK293 Cells ,Ubiquitin ligases ,Mutation ,Proteolysis ,Degron ,HeLa Cells - Abstract
Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis., The impact of genetic fusions on degrons, which are motifs for ubiquitin-mediated protein degradation, has not been fully explored. Here, the authors analyse fusion genes affecting degrons in pan-cancer genomics data, validate their functional impact and find enrichment for both internal and C-terminal degron losses.
- Published
- 2021
44. Structure of the human SAGA coactivator complex
- Author
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Meagan N. Esbin, Robert Tjian, Dominik A Herbst, Gina M. Dailey, Eva Nogales, Qianglin Fang, Xavier Darzacq, Robert K. Louder, and Claire Dugast-Darzacq
- Subjects
Transcription, Genetic ,Protein Conformation ,Transcriptional regulatory elements ,Medical and Health Sciences ,Structural Biology ,Regulatory Elements, Transcriptional ,Promoter Regions, Genetic ,Histone Acetyltransferases ,Regulation of gene expression ,Tumor ,Adaptor Proteins ,Nuclear Proteins ,Biological Sciences ,Chromatin ,SAGA complex ,RNA splicing ,Transcriptional ,Epigenetics ,Transcription ,Saccharomyces cerevisiae Proteins ,Phytic Acid ,1.1 Normal biological development and functioning ,Protein subunit ,Biophysics ,Computational biology ,Saccharomyces cerevisiae ,Biology ,Article ,Cell Line ,Promoter Regions ,Genetic ,Underpinning research ,Cell Line, Tumor ,Coactivator ,Genetics ,Humans ,Binding site ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Binding Sites ,Cryoelectron Microscopy ,Signal Transducing ,Regulatory Elements ,Yeast ,Gene Expression Regulation ,Hela Cells ,Chemical Sciences ,Saccharomycetales ,Chromatin modification ,Generic health relevance ,Developmental Biology ,HeLa Cells - Abstract
The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer., The cryo-EM structure of the human SAGA coactivator complex reveals high-resolution details of the core and TRRAP modules, providing insights on a metazoan-specific architecture and the structural basis for incorporation of the splicing module in mammalian cells.
- Published
- 2021
45. Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites
- Author
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Yanyan Yang, Sizhong Wu, Jiao An, Mengdie Yin, Aziz Sancar, Maoxiang Qian, Guoliang Xu, Jiayong Yin, Christopher P. Selby, and Jinchuan Hu
- Subjects
Genome instability ,AcademicSubjects/SCI00010 ,Genome Integrity, Repair and Replication ,G-quadruplex ,medicine.disease_cause ,chemistry.chemical_compound ,Genetics ,medicine ,Deoxyguanosine ,Humans ,Promoter Regions, Genetic ,Polymerase ,biology ,Genome, Human ,Nucleotides ,Reproducibility of Results ,Promoter ,DNA ,Sequence Analysis, DNA ,G-Quadruplexes ,Oxidative Stress ,chemistry ,Biochemistry ,8-Hydroxy-2'-Deoxyguanosine ,biology.protein ,Feasibility Studies ,GC-content ,Oxidative stress ,Algorithms ,DNA Damage ,Genome-Wide Association Study ,HeLa Cells - Abstract
8-Oxo-7,8-dihydro-2′-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.
- Published
- 2021
46. Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent
- Author
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Liu-Fu Chun, Zhiyi Chen, and Feng Yang
- Subjects
Survivin ,Genetic enhancement ,Contrast Media ,Apoptosis ,Gene delivery ,Transfection ,Biochemistry ,HeLa ,Humans ,Polyethyleneimine ,Cytotoxicity ,Molecular Biology ,Liposome ,Microbubbles ,biology ,Chemistry ,General Medicine ,biology.organism_classification ,Lipids ,Molecular biology ,MicroRNAs ,Drug delivery ,Molecular Medicine ,HeLa Cells ,Plasmids - Abstract
Background: Non-viral delivery systems is a promising method for gene or drug delivery. Polyethyleneimine (PEI) is a double-edged sword. It internalizes itself into the cell through endocytosis and promotes gene transfer efficiency. However, the strong positive charge also makes PEI highly toxic to cells. Ultrasound-targeted microbubble destruction (UTMD) is a promising non-viral method for gene and drug delivery, but its efficiency still needs to be improved. Objective: The aim of this study was to explore a system that combines ultrasound with non-viral gene delivery for the treatment of cervical cancer HeLa cells. Methods: In this study, we synthesized a kind of cationic ultrasound contrast agent(CUCA) that the physical and chemical properties, gene carrying capacity and cytotoxicity were verified. On the basis of previous studies, we further optimized the following transfusion parameters including ultrasound parameters, microbubble concentration, plasmid concentration, cell density and other parameters. The experiment was designed to compare the following six groups: (1) Plasmid group (P group), plasmid 15 μg; (2) PEI + plasmid group (PEI + P group),1 μl of PEI containing 10 nmol nitrogen and 1 μg of DNA containing 3 nmol phosphate for a PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection; (3) Ultrasound + plasmid group (US + P), plasmid 15 μg; (4) Ultrasound + cationic liposomal ultrasound contrast agent + plasmid group (UTMD + P group), plasmid 15 μg and cationic liposomal ultrasound contrast agent 5%; (5) Ultrasound + cationic liposomal ultrasound contrast agent + PEI + plasmid group (UTMD + PEI + P group), PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection and cationic liposomal ultrasound contrast agent 5%; and (6) Blank group, no treatment), The influence on Hela cells was observed under microscope, the efficiency of apoptosis was measured by flow cytometry, and cell viability was tested in CCK 8. Results: The optimized transfection parameters can improve the transfection efficiency of ultrasound combined with C-UCA to a certain extent, but its transfection efficiency is still lower than that of branched polyethyleneimine (bPEI) 25 kDa. By investigating the effect of HeLa cells apoptosis induced by UTMD in combination with PEI mediated survivin miRNA, we found that both PEI alone and ultrasound in combination with CUCA were able to transfect cells with survivin miRNA to effectively induce HeLa cell apoptosis. However, the synergistic effect between the two methods was not significant. Conclusion: In contrast, the combined use of ultrasound, C-UCA and PEI may significantly reduce the transfection efficiency of UTMD and PEI, and the specific mechanism remains to be further studied.
- Published
- 2021
47. miR-514a-3p: a novel SHP-2 regulatory miRNA that modulates human cytotrophoblast proliferation
- Author
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Karen Forbes, Rachel Quilang, and Sylvia Lui
- Subjects
animal structures ,placenta ,miR-758 ,medicine.medical_treatment ,proliferation ,miR-514 ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,Protein tyrosine phosphatase ,Biology ,PTPN11 ,Endocrinology ,Pregnancy ,microRNA ,medicine ,Humans ,IGF ,Molecular Biology ,miRNA ,Cell Proliferation ,Messenger RNA ,Cytotrophoblast ,Growth factor ,Research ,Trophoblast ,Transfection ,trophoblast ,Cell biology ,Trophoblasts ,MicroRNAs ,medicine.anatomical_structure ,embryonic structures ,SHP-2 ,Female ,Proto-oncogene tyrosine-protein kinase Src ,HeLa Cells - Abstract
Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP-2), encoded by the PTPN11 gene, forms a central component of multiple signalling pathways and is required for insulin-like growth factor (IGF)-induced placental growth. Altered expression of SHP-2 is associated with aberrant placental and fetal growth indicating that drugs modulating SHP-2 expression may improve adverse pregnancy outcome associated with altered placental growth. We have previously demonstrated that placental PTPN11/SHP-2 expression is controlled by miRNAs. SHP-2 regulatory miRNAs may have therapeutic potential; however, the individual miRNA(s) that regulate SHP-2 expression in the placenta remain to be established. We performed in silico analysis of 3’UTR target prediction databases to identify libraries of Hela cells transfected with individual miRNA mimetics, enriched in potential SHP-2 regulatory miRNAs. Analysis of PTPN11 levels by quantitative (q) PCR revealed that miR-758-3p increased, while miR-514a-3p reduced PTPN11 expression. The expression of miR-514a-3p and miR-758-3p within the human placenta was confirmed by qPCR; miR-514a-3p (but not miR-758-3p) levels inversely correlated with PTPN11 expression. To assess the interaction between these miRNAs and PTPN11/SHP-2, specific mimetics were transfected into first-trimester human placental explants and then cultured for up to 4 days. Overexpression of miR-514a-3p, but not miR-758-3p, significantly reduced PTPN11 and SHP-2 expression. microRNA-ribonucleoprotein complex (miRNP)-associated mRNA assays confirmed that this interaction was direct. miR-514a-3p overexpression attenuated IGF-I-induced trophoblast proliferation (BrdU incorporation). miR-758-3p did not alter trophoblast proliferation. These data demonstrate that by modulating SHP-2 expression, miR-514a-3p is a novel regulator of IGF signalling and proliferation in the human placenta and may have therapeutic potential in pregnancies complicated by altered placental growth.
- Published
- 2021
48. Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos
- Author
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Yan Xu, Junjiu Huang, Tianqi Cao, Jing Guo, Huan Zhao, Lizi Cheng, Yanwen Xu, Shan Jiang, Xiufeng Lin, Weifen Deng, and Min Gao
- Subjects
Adult ,Mutant ,Embryonic Development ,Polar Bodies ,Biology ,Transfection ,medicine.disease_cause ,Microtubules ,Mice ,Polar body ,Meiosis ,Tubulin ,Microtubule ,medicine ,Animals ,Humans ,Gene ,Microinjection ,Mutation ,Obstetrics and Gynecology ,Molecular biology ,Pedigree ,Reproductive Medicine ,Fertilization ,Oocytes ,Female ,Infertility, Female ,HeLa Cells ,Developmental Biology - Abstract
Research question How can the effect of genetic mutations that may cause primary female infertility be evaluated? Design Patients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes. Results A maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%). Conclusion Two TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.
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- 2021
49. Two New Cytotoxic Maytansinoids Targeting Tubulin from Trewia nudiflora
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Ya-Nan Li, Yubo Zhou, Jia Li, Jia-Nan Li, Ai-Jun Hou, Ming-Jun Cui, Chun Lei, and Kai-Cong Fu
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Dried fruit ,Pharmaceutical Science ,Antineoplastic Agents ,Analytical Chemistry ,HeLa ,Tubulin ,Cell Line, Tumor ,Drug Discovery ,Humans ,Cytotoxicity ,Pharmacology ,Molecular Structure ,biology ,Chemistry ,Organic Chemistry ,Euphorbiaceae ,Absolute configuration ,biology.organism_classification ,In vitro ,Complementary and alternative medicine ,Biochemistry ,Cell culture ,MCF-7 Cells ,biology.protein ,Molecular Medicine ,HeLa Cells - Abstract
Two new maytansinoids, N-methyltreflorine (1) and methyltrewiasine (2), were isolated from the dried fruits of Trewia nudiflora, together with three known congeners (3 – 5). Their structures were elucidated by spectroscopic methods, and the absolute configuration of 1 and 2 was determined by X-ray crystallographic analysis. Compounds 1 – 5 exhibited strong cytotoxicity against human tumor cell lines, including HeLa, MV-4 – 11, and MCF-7, with IC50 values ranging from 0.12 to 11 nM. Compounds 1 and 4 also showed inhibitory activity against the MCF-7/ADR cell line with IC50 values of 13 and 28 nM, respectively. Compounds 1 and 2 significantly inhibited tubulin polymerization in vitro with IC50 values of 3.6 and 3.2 µM, respectively.
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- 2021
50. Pharmacokinetics and Metabolites of 12 Bioactive Polymethoxyflavones in Rat Plasma
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Hu Yuan, Hongping Chen, Youping Liu, Qiang You, Hai-yan Ding, and Dan Li
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biology ,Chemistry ,Metabolite ,Hydroxy group ,Hep G2 Cells ,General Chemistry ,Health benefits ,Pharmacology ,Flavones ,HCT116 Cells ,biology.organism_classification ,Rats ,HeLa ,Structure-Activity Relationship ,chemistry.chemical_compound ,Pharmacokinetics ,A549 Cells ,Animals ,Humans ,Cancer cell lines ,General Agricultural and Biological Sciences ,Cytotoxicity ,HeLa Cells - Abstract
Polymethoxyflavones (PMFs) are a subgroup of flavonoids possessing various health benefits. 3,5,7,4'-Tetramethoxyflavone (1), 5,6,7,4'-tetramethylflavone (2), 3,7,3',4'-tetramethoxyflavone (3), 5,7,3',4'-tetramethoxyflavone (4), 5-hydroxy-3,7,2',4'-tetramethoxyflavone (5), 3,5,7,2',4'-pentamethoxyflavone (6), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (7), 3-hydroxy-5,7,3',4'-tetramethylflavone (8), 3,5,7,3',4'-pentamethoxyflavone (9), 5-hydroxy-3,7,3',4',5'-pentamethoxyflavone (10), 3-hydroxy-5,7,3',4',5'-pentamethoxyflavone (11), and 3,5,7,3',4',5'-hexamethoxylflavone (12) were 12 bioactive and available PMFs. The aim of this study was to investigate the pharmacokinetic, metabolite, and antitumor activities as well as the structure-pharmacokinetic-antitumor activity relationships of these 12 PMFs to facilitate further studies of their medicinal potentials. The cytotoxicity of PMFs with a hydroxy group toward HeLa, A549, HepG2, and HCT116 cancer cell lines was generally significantly more potent than that of PMFs without a hydroxy group. Compounds 5, 7, 8, 10, and 11 were all undetectable in rat plasma, while compounds 1-4, 6, 9, and 12 were detectable. Both the number and position of hydroxy and methoxy groups played an important role in modulating PMF pharmacokinetics and metabolites.
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- 2021
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