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Molecular Biology: Feature Papers
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Molecular Biology: Feature Papers

A topical collection in Biomolecules (ISSN 2218-273X). This collection belongs to the section "Molecular Biology".

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Editor

Prof. Dr. Mark S. Johnson

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Collection Editor
Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, 20520 Turku, Finland
Interests: structure-function relationships; structural bioinformatics; structure modeling; ligand binding and function; protein evolution; effects of mutations; EGFR family of tyrosine kinases; integrins; GPCRs; PDR-like family of transcription factors; calcium binding proteins; proteases
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Topical Collection Information

Dear Colleagues,

This Topical Collection entitled “Molecular Biology: Feature Papers” aims to collect high-quality research articles, review articles, and communications on all aspects of molecular biology. It is dedicated to recent advances in the broad research area of molecular biology; is inclusive of papers focused on wet-lab experimental studies, computational studies, and their synergistic combination; and is comprised of a selection of exclusive papers from the Editorial Board Members (EBMs) of the Molecular Biology Section, as well as invited papers from relevant experts. We also welcome senior experts in the field to make contributions to this Topical Collection. Kindly note that all invited papers will be published online free of charge once accepted. We aim to represent our section as an attractive open-access publishing platform for molecular biology research.

Prof. Dr. Mark Johnson
Collection Editor

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Published Papers (25 papers)

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2023

Jump to: 2022, 2021, 2020

18 pages, 7637 KiB  
Article
A Structural Model for the Core Nup358-BicD2 Interface
by James M. Gibson, Xiaoxin Zhao, M. Yusuf Ali, Sozanne R. Solmaz and Chunyu Wang
Biomolecules 2023, 13(10), 1445; https://doi.org/10.3390/biom13101445 - 26 Sep 2023
Viewed by 1247
Abstract
Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct [...] Read more.
Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a “cargo recognition α-helix” upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development. Full article
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25 pages, 4351 KiB  
Article
Integrated Metabolomic and Transcriptomic Analysis of the Quinoa Seedling Response to High Relative Humidity Stress
by Xinyi Li, Ping Zhang, Jia Liu, Hongxin Wang, Junna Liu, Hanxue Li, Heng Xie, Qianchao Wang, Li Li, Shan Zhang, Liubin Huang, Chenghong Liu and Peng Qin
Biomolecules 2023, 13(9), 1352; https://doi.org/10.3390/biom13091352 - 05 Sep 2023
Viewed by 962
Abstract
Quinoa is of great interest because it is cold- and drought-resistant; however, little research has been performed on quinoa under high relative humidity (RH) stress. In this study, quinoa seedlings of a highly HR-resistant variety (“Dianli-439”) and a sensitive variety (“Dianli-969”) were subjected [...] Read more.
Quinoa is of great interest because it is cold- and drought-resistant; however, little research has been performed on quinoa under high relative humidity (RH) stress. In this study, quinoa seedlings of a highly HR-resistant variety (“Dianli-439”) and a sensitive variety (“Dianli-969”) were subjected to morphological and physiological measurements and metabolome and transcriptome analyses to investigate their response to high RH stress. In total, 1060 metabolites were detected, and lipids and flavonoids were the most abundant, with 173 and 167 metabolites, respectively. In total, 13,095 differentially expressed genes were identified, and the results showed that abscisic acid, auxin, and jasmonic-acid-related genes involved in plant hormone signaling may be involved in the response of quinoa seedlings to high RH stress. The analysis of the transcription factors revealed that the AP2/ERF family may also play an important role in the response to high RH stress. We identified the possible regulatory mechanisms of the hormone signaling pathways under high RH stress. Our findings can provide a basis for the selection and identification of highly resistant quinoa varieties and the screening of the metabolite-synthesis- and gene-regulation-related mechanisms in quinoa in response to RH stress. Full article
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18 pages, 3188 KiB  
Article
Molecular Influence of the ATM Protein in the Treatment of Human Cells with Different Radioprotective Drugs: Comparisons between Antioxidative and Pro-Episkevic Strategies
by Juliette Restier-Verlet, Michel Drouet, Pauline Pras, Mélanie L. Ferlazzo, Adeline Granzotto, Laurène Sonzogni, Joëlle Al-Choboq, Laura El Nachef, Sabine François, Michel Bourguignon and Nicolas Foray
Biomolecules 2023, 13(3), 524; https://doi.org/10.3390/biom13030524 - 13 Mar 2023
Cited by 1 | Viewed by 1208
Abstract
The radiation protection strategy with chemical agents has long been based on an antioxidative approach consisting in reducing the number of radical oxygen and nitrogen species responsible for the formation of the radiation-induced (RI) DNA damage, notably the DNA double-strand breaks (DSB), whose [...] Read more.
The radiation protection strategy with chemical agents has long been based on an antioxidative approach consisting in reducing the number of radical oxygen and nitrogen species responsible for the formation of the radiation-induced (RI) DNA damage, notably the DNA double-strand breaks (DSB), whose subset participates in the RI lethal effect as unrepairable damage. Conversely, a DSB repair-stimulating strategy that may be called the “pro-episkevic” approach (from the ancient Greek episkeve, meaning repair) can be proposed. The pro-episkevic approach directly derives from a mechanistic model based on the RI nucleoshuttling of the ATM protein (RIANS) and contributes to increase the number of DSB managed by NHEJ, the most predominant DSB repair and signaling pathway in mammalians. Here, three radioresistant and three radiosensitive human fibroblast cell lines were pretreated with antioxidative agents (N-acetylcysteine or amifostine) or to two pro-episkevic agents (zoledronate or pravastatin or both (ZOPRA)) before X-ray irradiation. The fate of the RI DSB was analyzed by using γH2AX and pATM immunofluorescence. While amifostine pretreatment appeared to be the most efficient antioxidative process, ZOPRA shows the most powerful radiation protection, suggesting that the pro-episkevic strategy may be an alternative to the antioxidative one. Additional investigations are needed to develop some new drugs that may elicit both antioxidative and pro-episkevic properties and to quantify the radiation protection action of both types of drugs applied concomitantly. Full article
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14 pages, 1489 KiB  
Article
Various Expressions of PIK3C2A and TXNIP Genes and Their Potential Role as Independent Risk Factors for Chronic Stable Angina and Acute Coronary Syndrome
by Shimaa E. Soliman, Mai A. H. Abouelenin, Neven I. Samy, Marwa M. Omar and Abeer A. Alrefai
Biomolecules 2023, 13(2), 302; https://doi.org/10.3390/biom13020302 - 06 Feb 2023
Cited by 2 | Viewed by 1294
Abstract
Background and Aim: Genetic factors play a significant role in the onset and progression of coronary artery disease (CAD). PIK3C2A may contribute to the development of acute coronary syndrome (ACS) by affecting blood glucose levels and oxidative stress. The expression levels of TXNIP [...] Read more.
Background and Aim: Genetic factors play a significant role in the onset and progression of coronary artery disease (CAD). PIK3C2A may contribute to the development of acute coronary syndrome (ACS) by affecting blood glucose levels and oxidative stress. The expression levels of TXNIP were significantly higher in patients with unstable angina pectoris. However, the situation is different in ACS. In the current study, we aim to investigate the role of PIK3C2A and TXNIP as independent risk factors for chronic stable angina (CSA) and ACS. Subjects and Methods: This study involved 215 subjects (60 patients with CSA, 55 patients with ACS, and 100 controls). All subjects were exposed for assaying gene expressions of PIK3C2A and TXNIP by quantitative real-time polymerase chain reaction. Results: It was found that TXNIP was upregulated, whereas PIK3C2A was downregulated in patients with CAD compared to the control group. PIK3C2A was significantly downregulated in patients with ACS compared to that in patients with CSA (p < 0.001), but TXNIP was not (p = 0.7). TXNIP was significantly upregulated in STEMI-ACS patients compared to CSA (p = 0.045) and NSTEMI ACS (p = 0.046), among non-diabetic (p = 0.023) smokers (p = 0.036) with hypertension (p = 0.005) and hypercholesterolemia (p = 0.001). ROC (receiver operating characteristic) curve analysis revealed that PIK3C2A (0.981; p < 0.001; 98.18) was the most sensitive mRNA for discriminating ACS from control, followed by TXNIP (0.775; p < 0.001; 70.91). However, for discriminating ACS from CSA combined mRNAs, (PIK3C2A + TXNIP) (0.893; p < 0.001; 98.18) and PIK3C2A (0.892; p < 0.001; 81.82) are promising biomarkers. On the other hand, the most sensitive mRNA for differentiating CSA from control is mRNAs (PIK3C2A + TXNIP) (0.963; p < 0.001; 95), then TXINP (81.3; p < 0.001; 93.33), and finally, PIK3C2A (0.782; p < 0.001; 81.67). In the multivariate regression model, PIK3C2A ((p = 0.002), 0.118 (0.031–0.445)) and smoking status ((p = 0.034); 0.151 (0.026–0.866)) were independent variables for ACS. Moreover, PIK3C2A ((p < 0.013); 0.706 (0.614–0.812)), Hb ((p = 0.013); 0.525 (0.317–0.871)), and total cholesterol ((p = 0.04); 0.865 (0.784–0.955)) were significantly (p < 0.05) and independently related to the prognosis of CSA. Furthermore, PIK3C2A ((p = 0.002), 0.923 (0.877–0.971)), TXNIP ((p = 0.001); 2.809 (1.558–5.064)) the body weight ((p = 0.033); 1.254 (1.018–1.544)) were independently associated with CSA. Conclusions: Our study concluded that the dysregulated mRNA PIK3C2A and TXNIP gene expressions may be useful in diagnosis of CAD and prediction of ACS development. Full article
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2022

Jump to: 2023, 2021, 2020

11 pages, 1985 KiB  
Article
Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases
by Wataru Aiba, Takamitsu Amai, Mitsuyoshi Ueda and Kouichi Kuroda
Biomolecules 2022, 12(11), 1621; https://doi.org/10.3390/biom12111621 - 03 Nov 2022
Cited by 2 | Viewed by 1985
Abstract
In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we [...] Read more.
In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing. Full article
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9 pages, 2168 KiB  
Article
Proline-Rich Region II (PRR2) Plays an Important Role in Tau–Glycan Interaction: An NMR Study
by Anqesha Murray, Lufeng Yan, James M. Gibson, Jian Liu, David Eliezer, Guy Lippens, Fuming Zhang, Robert J. Linhardt, Jing Zhao and Chunyu Wang
Biomolecules 2022, 12(11), 1573; https://doi.org/10.3390/biom12111573 - 27 Oct 2022
Cited by 1 | Viewed by 2078
Abstract
(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer’s disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR [...] Read more.
(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer’s disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau–heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*–Arixtra interaction had a much stronger binding affinity (0.37–0.67 mM) than that of tau R2*–Arixtra (1.90–5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau–heparin and tau–HS interaction. Full article
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25 pages, 3082 KiB  
Review
Activation and Pharmacological Regulation of Inflammasomes
by Chen Chen and Pinglong Xu
Biomolecules 2022, 12(7), 1005; https://doi.org/10.3390/biom12071005 - 20 Jul 2022
Cited by 19 | Viewed by 4647
Abstract
Inflammasomes are intracellular signaling complexes of the innate immune system, which is part of the response to exogenous pathogens or physiological aberration. The multiprotein complexes mainly consist of sensor proteins, adaptors, and pro-caspase-1. The assembly of the inflammasome upon extracellular and intracellular cues [...] Read more.
Inflammasomes are intracellular signaling complexes of the innate immune system, which is part of the response to exogenous pathogens or physiological aberration. The multiprotein complexes mainly consist of sensor proteins, adaptors, and pro-caspase-1. The assembly of the inflammasome upon extracellular and intracellular cues drives the activation of caspase-1, which processes pro-inflammatory cytokines IL-1β and IL-18 to maturation and gasdermin-D for pore formation, leading to pyroptosis and cytokine release. Inflammasome signaling functions in numerous infectious or sterile inflammatory diseases, including inherited autoinflammatory diseases, metabolic disorders, cardiovascular diseases, cancers, neurodegenerative disorders, and COVID-19. In this review, we summarized current ideas on the organization and activation of inflammasomes, with details on the molecular mechanisms, regulations, and interventions. The recent developments of pharmacological strategies targeting inflammasomes as disease therapeutics were also covered. Full article
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17 pages, 2547 KiB  
Article
Transcriptomic and Metabolomic Analysis of the Response of Quinoa Seedlings to Low Temperatures
by Heng Xie, Qianchao Wang, Ping Zhang, Xuesong Zhang, Tingzhi Huang, Yirui Guo, Junna Liu, Li Li, Hanxue Li and Peng Qin
Biomolecules 2022, 12(7), 977; https://doi.org/10.3390/biom12070977 - 12 Jul 2022
Cited by 6 | Viewed by 2503
Abstract
Quinoa, a cool-weather high-altitude crop, is susceptible to low-temperature stress throughout its reproductive phase. Herein, we performed broadly targeted metabolic profiling of quinoa seedlings to explore the metabolites’ dynamics in response to low-temperature stress and transcriptome analysis to determine the underlying genetic mechanisms. [...] Read more.
Quinoa, a cool-weather high-altitude crop, is susceptible to low-temperature stress throughout its reproductive phase. Herein, we performed broadly targeted metabolic profiling of quinoa seedlings to explore the metabolites’ dynamics in response to low-temperature stress and transcriptome analysis to determine the underlying genetic mechanisms. Two variants, namely, Dian Quinoa 2324 and Dian Quinoa 281, were exposed to temperatures of −2, 5, and 22 °C. A total of 794 metabolites were detected; 52,845 genes, including 6628 novel genes, were annotated using UPLC-MS/MS analysis and the Illumina HiSeq system. Combined with morphological indicators to resolve the mechanism underlying quinoa seedling response to low-temperature stress, the molecular mechanisms of quinoa changed considerably based on temperature exposure. Soluble sugars heavily accumulated in plants with cold damage and changes in regulatory networks under freeze damage, such as the upregulation of α-linolenic acid metabolism and a reduction in energy substrates, may explain the spatial patterns of biosynthesis and accumulation of these metabolites. Genes that are actively expressed during cold responses, as revealed by co-expression analyses, may be involved in the regulation thereof. These results provide insights into the metabolic factors in quinoa under low-temperature stress and provide a reference for the screening of quinoa varieties resistant to low temperature. Full article
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15 pages, 1571 KiB  
Review
Potential to Eradicate Cancer Stemness by Targeting Cell Surface GRP78
by Hsin-Ying Chen and Ann-Joy Cheng
Biomolecules 2022, 12(7), 941; https://doi.org/10.3390/biom12070941 - 05 Jul 2022
Cited by 1 | Viewed by 2078
Abstract
Cancer stemness is proposed to be the main cause of metastasis and tumor relapse after conventional therapy due to the main properties of cancer stem cells. These include unlimited self-renewal, the low percentage in a cell population, asymmetric/symmetric cell division, and the hypothetical [...] Read more.
Cancer stemness is proposed to be the main cause of metastasis and tumor relapse after conventional therapy due to the main properties of cancer stem cells. These include unlimited self-renewal, the low percentage in a cell population, asymmetric/symmetric cell division, and the hypothetical different nature for absorbing external substances. As the mechanism of how cancer stemness is maintained remains unknown, further investigation into the basic features of cancer stemness is required. Many articles demonstrated that glucose-regulated protein 78 (GRP78) plays a key role in cancer stemness, suggesting that this molecule is feasible for targeting cancer stem cells. This review summarizes the history of finding cancer stem cells, as well as the functions of GRP78 in cancer stemness, for discussing the possibility of targeting GRP78 to eradicate cancer stemness. Full article
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15 pages, 986 KiB  
Review
Effects of Glucose Metabolism, Lipid Metabolism, and Glutamine Metabolism on Tumor Microenvironment and Clinical Implications
by Longfei Zhu, Xuanyu Zhu and Yan Wu
Biomolecules 2022, 12(4), 580; https://doi.org/10.3390/biom12040580 - 14 Apr 2022
Cited by 38 | Viewed by 5739
Abstract
In recent years, an increasingly more in depth understanding of tumor metabolism in tumorigenesis, tumor growth, metastasis, and prognosis has been achieved. The broad heterogeneity in tumor tissue is the critical factor affecting the outcome of tumor treatment. Metabolic heterogeneity is not only [...] Read more.
In recent years, an increasingly more in depth understanding of tumor metabolism in tumorigenesis, tumor growth, metastasis, and prognosis has been achieved. The broad heterogeneity in tumor tissue is the critical factor affecting the outcome of tumor treatment. Metabolic heterogeneity is not only found in tumor cells but also in their surrounding immune and stromal cells; for example, many suppressor cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tumor-associated T-lymphocytes. Abnormalities in metabolism often lead to short survival or resistance to antitumor therapy, e.g., chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Using the metabolic characteristics of the tumor microenvironment to identify and treat cancer has become a great research hotspot. This review systematically addresses the impacts of metabolism on tumor cells and effector cells and represents recent research advances of metabolic effects on other cells in the tumor microenvironment. Finally, we introduce some applications of metabolic features in clinical oncology. Full article
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28 pages, 14707 KiB  
Review
DNA Methylation Malleability and Dysregulation in Cancer Progression: Understanding the Role of PARP1
by Rakesh Srivastava and Niraj Lodhi
Biomolecules 2022, 12(3), 417; https://doi.org/10.3390/biom12030417 - 08 Mar 2022
Cited by 7 | Viewed by 4322
Abstract
Mammalian genomic DNA methylation represents a key epigenetic modification and its dynamic regulation that fine-tunes the gene expression of multiple pathways during development. It maintains the gene expression of one generation of cells; particularly, the mitotic inheritance of gene-expression patterns makes it the [...] Read more.
Mammalian genomic DNA methylation represents a key epigenetic modification and its dynamic regulation that fine-tunes the gene expression of multiple pathways during development. It maintains the gene expression of one generation of cells; particularly, the mitotic inheritance of gene-expression patterns makes it the key governing mechanism of epigenetic change to the next generation of cells. Convincing evidence from recent discoveries suggests that the dynamic regulation of DNA methylation is accomplished by the enzymatic action of TET dioxygenase, which oxidizes the methyl group of cytosine and activates transcription. As a result of aberrant DNA modifications, genes are improperly activated or inhibited in the inappropriate cellular context, contributing to a plethora of inheritable diseases, including cancer. We outline recent advancements in understanding how DNA modifications contribute to tumor suppressor gene silencing or oncogenic-gene stimulation, as well as dysregulation of DNA methylation in cancer progression. In addition, we emphasize the function of PARP1 enzymatic activity or inhibition in the maintenance of DNA methylation dysregulation. In the context of cancer remediation, the impact of DNA methylation and PARP1 pharmacological inhibitors, and their relevance as a combination therapy are highlighted. Full article
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13 pages, 2098 KiB  
Article
Human β-Defensin 2 (HBD-2) Displays Oncolytic Activity but Does Not Affect Tumour Cell Migration
by Guneet K. Bindra, Scott A. Williams, Fung T. Lay, Amy A. Baxter, Ivan K. H. Poon, Mark D. Hulett and Thanh Kha Phan
Biomolecules 2022, 12(2), 264; https://doi.org/10.3390/biom12020264 - 06 Feb 2022
Cited by 9 | Viewed by 3790
Abstract
Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. [...] Read more.
Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development. Full article
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10 pages, 1536 KiB  
Article
PEPscan: A Broad Spectrum Approach for the Characterization of Protein-Binder Interactions?
by Angelita Rebollo, Louise Fliedel and Pierre Tuffery
Biomolecules 2022, 12(2), 178; https://doi.org/10.3390/biom12020178 - 21 Jan 2022
Cited by 1 | Viewed by 2318
Abstract
In a previous study, we have shown that PEPscan can provide a cheap and rapid means to identify candidate interfering peptides (IPs), i.e., peptides able to disrupt a target protein-protein interaction. PEPscan was shown to be effective in identifying a limited number of [...] Read more.
In a previous study, we have shown that PEPscan can provide a cheap and rapid means to identify candidate interfering peptides (IPs), i.e., peptides able to disrupt a target protein-protein interaction. PEPscan was shown to be effective in identifying a limited number of candidate IPs specific to the target interaction. Here, we investigate the results of 14 new PEPscan experiments for protein complexes of known 3D structures. We show that for almost all complexes, PEPscan is able to identify candidate IPs that are located at the protein-protein interface. The information it provides about the binding site seems, however, too ambiguous to be exploited in a simple manner to assist the modeling of protein complexes. Moreover, these candidates are associated with false positives. For these, we suggest they could correspond to non-specific binders, which leaves room for further optimization of the PEPscan protocol. Another unexpected advance comes from the observation of the applicability of PEPscan for polysaccharides and labeled peptides, suggesting that PEPscan could become a large spectrum approach to investigate protein-binder interactions, the binder not necessarily being a protein. Full article
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2021

Jump to: 2023, 2022, 2020

17 pages, 3200 KiB  
Article
Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models
by Simona Serratì, Antonio Palazzo, Annamaria Lapenna, Helena Mateos, Antonia Mallardi, René Massimiliano Marsano, Alessandra Quarta, Mario Del Rosso and Amalia Azzariti
Biomolecules 2021, 11(12), 1857; https://doi.org/10.3390/biom11121857 - 10 Dec 2021
Cited by 2 | Viewed by 2740
Abstract
The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less [...] Read more.
The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation. Full article
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23 pages, 5766 KiB  
Article
Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus
by Alexander A. Charbonneau, Debra M. Eckert, Colin C. Gauvin, Nathanael G. Lintner and C. Martin Lawrence
Biomolecules 2021, 11(12), 1852; https://doi.org/10.3390/biom11121852 - 09 Dec 2021
Cited by 5 | Viewed by 2784
Abstract
Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here [...] Read more.
Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating “ring nuclease” activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response. Full article
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15 pages, 4586 KiB  
Article
Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins
by Guangbo Yan, Xia Li, Jun Yang, Zhongchen Li, Jia Hou, Ben Rao, Yong Hu, Lixin Ma and Yaping Wang
Biomolecules 2021, 11(11), 1706; https://doi.org/10.3390/biom11111706 - 17 Nov 2021
Cited by 7 | Viewed by 2296
Abstract
Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies [...] Read more.
Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins. Full article
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10 pages, 2097 KiB  
Article
Sgt1 Regulates α-Synuclein Subcellular Localization and Expression of Parkinson’s Disease Related Genes, PINK1 and PARK9
by Anastasiia Bohush, Agnieszka Góral, Małgorzata Sierant, Barbara Nawrot, Wiesława Leśniak and Anna Filipek
Biomolecules 2021, 11(11), 1675; https://doi.org/10.3390/biom11111675 - 11 Nov 2021
Cited by 1 | Viewed by 1867
Abstract
The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as [...] Read more.
The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD). In the present work we have found that SGT1 protected cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. To gain more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in HEK293 cells with an altered SGT1 level. By immunofluorescent staining we have found that in HEK293 cells overexpressing SGT1 α-synuclein was mainly localized in the cytoplasm while in control cells it was present in the nucleus. Accordingly, when SGT1 expression was silenced, α-synuclein was predominantly present in the nucleus. These results were then confirmed by subcellular fractionation and Western blot analysis. Moreover, we have found that altered level of SGT1 in HEK293 cells influenced the expression of PD related genes, PINK1 and PARK9. Altogether, our results point to SGT1 as an important factor that might be involved in the pathogenesis of Parkinson’s disease (PD). Full article
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17 pages, 3374 KiB  
Article
Comprehensive Atlas of the Myelin Basic Protein Interaction Landscape
by Evgeniya V. Smirnova, Tatiana V. Rakitina, Rustam H. Ziganshin, Georgij P. Arapidi, George A. Saratov, Anna A. Kudriaeva and Alexey A. Belogurov
Biomolecules 2021, 11(11), 1628; https://doi.org/10.3390/biom11111628 - 03 Nov 2021
Cited by 11 | Viewed by 3224
Abstract
Intrinsically disordered myelin basic protein (MBP) is one of the key autoantigens in autoimmune neurodegeneration and multiple sclerosis particularly. MBP is highly positively charged and lacks distinct structure in solution and therefore its intracellular partners are still mostly enigmatic. Here we used combination [...] Read more.
Intrinsically disordered myelin basic protein (MBP) is one of the key autoantigens in autoimmune neurodegeneration and multiple sclerosis particularly. MBP is highly positively charged and lacks distinct structure in solution and therefore its intracellular partners are still mostly enigmatic. Here we used combination of formaldehyde-induced cross-linking followed by immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the interaction network of MBP in mammalian cells and provide the list of potential MBP interacting proteins. Our data suggest that the largest group of MBP-interacting proteins belongs to cellular proteins involved in the protein translation machinery, as well as in the spatial and temporal regulation of translation. MBP interacts with core ribosomal proteins, RNA helicase Ddx28 and RNA-binding proteins STAU1, TDP-43, ADAR-1 and hnRNP A0, which are involved in various stages of RNA biogenesis and processing, including specific maintaining MBP-coding mRNA. Among MBP partners we identified CTNND1, which has previously been shown to be necessary for myelinating Schwann cells for cell-cell interactions and the formation of a normal myelin sheath. MBP binds proteins MAGEB2/D2 associated with neurotrophin receptor p75NTR, involved in pathways that promote neuronal survival and neuronal death. Finally, we observed that MBP interacts with RNF40–a component of heterotetrameric Rnf40/Rnf20 E3 ligase complex, recruited by Egr2, which is the central transcriptional regulator of peripheral myelination. Concluding, our data suggest that MBP may be more actively involved in myelination not only as a main building block but also as a self-regulating element. Full article
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16 pages, 8167 KiB  
Article
Puerarin Attenuates Cadmium-Induced Neuronal Injury via Stimulating Cadmium Excretion, Inhibiting Oxidative Stress and Apoptosis
by Shuangquan Wen, Li Wang, Hui Zou, Jianhong Gu, Ruilong Song, Jianchun Bian, Yan Yuan and Zongping Liu
Biomolecules 2021, 11(7), 978; https://doi.org/10.3390/biom11070978 - 02 Jul 2021
Cited by 14 | Viewed by 2115
Abstract
Cadmium (Cd) is a potential pathogenic factor in the nervous system associated with various neurodegenerative disorders. Puerarin (Pur) is an isoflavone purified from the Chinese medical herb, kudzu root, and exhibits antioxidant and antiapoptotic properties in the brain. In this study, the detailed [...] Read more.
Cadmium (Cd) is a potential pathogenic factor in the nervous system associated with various neurodegenerative disorders. Puerarin (Pur) is an isoflavone purified from the Chinese medical herb, kudzu root, and exhibits antioxidant and antiapoptotic properties in the brain. In this study, the detailed mechanisms underlying the neuroprotective potential of Pur against Cd-induced neuronal injury was evaluated for the first time in vivo in a rat model and in vitro using primary rat cerebral cortical neurons. The results of the in vivo experiments showed that Pur ameliorated Cd-induced neuronal injury, reduced Cd levels in the cerebral cortices, and stimulated Cd excretion in Cd-treated rats. We also observed that the administration of Pur rescued Cd-induced oxidative stress, and attenuated Cd-induced apoptosis by concomitantly suppressing both the Fas/FasL and mitochondrial pathways in the cerebral cortical neurons of rats both in vivo and in vitro. Our results demonstrate that Pur exerted its neuroprotective effects by stimulating Cd excretion, ameliorating Cd-induced oxidative stress and apoptosis in rat cerebral cortical neurons. Full article
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13 pages, 2567 KiB  
Article
Pepscan Approach for the Identification of Protein–Protein Interfaces: Lessons from Experiment
by Angelita Rebollo, Eric Savier and Pierre Tuffery
Biomolecules 2021, 11(6), 772; https://doi.org/10.3390/biom11060772 - 21 May 2021
Cited by 3 | Viewed by 2156
Abstract
PEPscan is an old approach that has recently gained renewed interest for the identification of interfering peptides (IPs), i.e., peptides able to interfere with protein–protein interactions (PPIs). Its principle is to slice a protein sequence as a series of short overlapping peptides that [...] Read more.
PEPscan is an old approach that has recently gained renewed interest for the identification of interfering peptides (IPs), i.e., peptides able to interfere with protein–protein interactions (PPIs). Its principle is to slice a protein sequence as a series of short overlapping peptides that are synthesized on a peptide array and tested for their ability to bind a partner, with positive spots corresponding to candidate IPs. PEPscan has been applied with a rather large success in various contexts, but the structural determinants underlying this success remain obscure. Here, we analyze the results of 14 PEPscan experiments, and confront the in vitro results with the available structural information. PEPscan identifies candidate IPs in limited numbers that in all cases correspond to solvent-accessible regions of the structures, their location at the protein–protein interface remaining to be further demonstrated. A strong point of PEPscan seems to be its ability to identify specific IPs. IPs identified from the same protein differ depending on the target PPI, and correspond to patches not frequently involved in the interactions seen in the 3D structures available. Overall, PEPscan seems to provide a cheap and rapid manner to identify candidate IPs, that also comes with room for improvement. Full article
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35 pages, 6005 KiB  
Article
Molecular Targets and Biological Functions of cAMP Signaling in Arabidopsis
by Ruqiang Xu, Yanhui Guo, Song Peng, Jinrui Liu, Panyu Li, Wenjing Jia and Junheng Zhao
Biomolecules 2021, 11(5), 688; https://doi.org/10.3390/biom11050688 - 03 May 2021
Cited by 21 | Viewed by 4114
Abstract
Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. However, the mechanism of cAMP signaling in plants remains very poorly understood. Here, we employ the engineered activity of soluble adenylate cyclase to induce cellular cAMP elevation in Arabidopsis [...] Read more.
Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. However, the mechanism of cAMP signaling in plants remains very poorly understood. Here, we employ the engineered activity of soluble adenylate cyclase to induce cellular cAMP elevation in Arabidopsis thaliana plants and identify 427 cAMP-responsive genes (CRGs) through RNA-seq analysis. Induction of cellular cAMP elevation inhibits seed germination, disturbs phytohormone contents, promotes leaf senescence, impairs ethylene response, and compromises salt stress tolerance and pathogen resistance. A set of 62 transcription factors are among the CRGs, supporting a prominent role of cAMP in transcriptional regulation. The CRGs are significantly overrepresented in the pathways of plant hormone signal transduction, MAPK signaling, and diterpenoid biosynthesis, but they are also implicated in lipid, sugar, K+, nitrate signaling, and beyond. Our results provide a basic framework of cAMP signaling for the community to explore. The regulatory roles of cAMP signaling in plant plasticity are discussed. Full article
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16 pages, 2337 KiB  
Article
Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach
by Yan Liu, Yali Shen, Yang Song, Lei Xu, J. Jefferson P. Perry and Jiayu Liao
Biomolecules 2021, 11(5), 673; https://doi.org/10.3390/biom11050673 - 30 Apr 2021
Cited by 1 | Viewed by 2165
Abstract
Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack [...] Read more.
Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases. Full article
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10 pages, 1471 KiB  
Article
Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the −183 to −137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase
by Takahiro Nakayama, Toshiyuki Fukutomi, Yasuo Terao and Kimio Akagawa
Biomolecules 2021, 11(2), 146; https://doi.org/10.3390/biom11020146 - 23 Jan 2021
Cited by 4 | Viewed by 2226
Abstract
The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core [...] Read more.
The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC. Full article
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2020

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12 pages, 2256 KiB  
Article
Brain-Derived Neurotrophic Factor Regulates Ishikawa Cell Proliferation through the TrkB-ERK1/2 Signaling Pathway
by Maosheng Cao, Qiaoge Niu, XinYu Xiang, Chenfeng Yuan, Tariq Iqbal, Yuwen Huang, Meng Tian, Zijiao Zhao, Chunjin Li and Xu Zhou
Biomolecules 2020, 10(12), 1645; https://doi.org/10.3390/biom10121645 - 08 Dec 2020
Cited by 5 | Viewed by 4108
Abstract
(1) Background: Endometrial regulation is a necessary condition for maintaining normal uterine physiology, which is driven by many growth factors. Growth factors produced in the endometrium are thought to be related to the proliferation of endometrial cells induced by estradiol-17β (E2). [...] Read more.
(1) Background: Endometrial regulation is a necessary condition for maintaining normal uterine physiology, which is driven by many growth factors. Growth factors produced in the endometrium are thought to be related to the proliferation of endometrial cells induced by estradiol-17β (E2). In this study, we found that E2 can induce the secretion of brain-derived neurotrophic factor (BDNF) in Ishikawa cells (the cells of an endometrial cell line). Furthermore, Ishikawa cells were used in exploring the regulatory role of BDNF in endometrial cells and to clarify the potential mechanism. (2) Methods: Ishikawa cells were treated with different concentrations of BDNF (100, 200, 300, 400, and 500 ng/mL). The mRNA expression levels of various proliferation-related genes were detected through quantitative reverse transcription polymerase chain reaction, and the expression of various proliferation-related genes was detected by knocking out BDNF or inhibiting the binding of BDNF to its receptor TrkB. The expression levels of various proliferation-related genes were detected by performing Western blotting on the TrkB-ERK1/2 signaling pathway. (3) Results: Exogenous BDNF promoted the growth of the Ishikawa cells, but the knocking down of BDNF or the inhibition of TrkB reduced their growth. Meanwhile, BDNF enhanced cell viability and increased the expression of proliferation-related genes, including cyclin D1 and cyclin E2. More importantly, the BDNF-induced proliferation of the Ishikawa cells involved the ERK1/2 signaling pathway. (4) Conclusions: The stimulating effect of exogenous E2 on the expression of BDNF in the uterus and the action of BDNF promoted the proliferation of the Ishikawa cells through the TrkB-ERK1/2 signal pathway. Full article
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14 pages, 1952 KiB  
Article
Co-Application of Eugenol and QX-314 Elicits the Prolonged Blockade of Voltage-Gated Sodium Channels in Nociceptive Trigeminal Ganglion Neurons
by Sung-Min Hwang, Kihwan Lee, Sang-Taek Im, Eun Jin Go, Yong Ho Kim and Chul-Kyu Park
Biomolecules 2020, 10(11), 1513; https://doi.org/10.3390/biom10111513 - 05 Nov 2020
Cited by 11 | Viewed by 3745
Abstract
Local anesthetics (LAs) can completely block nociception by inhibiting voltage-gated sodium channels (VGSCs), and thus, blocking action potentials (APs) within sensory neurons. As one of the several LAs, eugenol is used for dental pain treatment. It reportedly features multiple functions in regulating diverse [...] Read more.
Local anesthetics (LAs) can completely block nociception by inhibiting voltage-gated sodium channels (VGSCs), and thus, blocking action potentials (APs) within sensory neurons. As one of the several LAs, eugenol is used for dental pain treatment. It reportedly features multiple functions in regulating diverse ion channels. This study aimed to investigate the long-lasting analgesic effect of eugenol alone, as well as that of the combination of eugenol as a noxious-heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channel agonist and a permanently charged sodium channel blocker (QX-314), on neuronal excitability in trigeminal ganglion (TG) neurons. Eugenol alone increased inward current in a dose-dependent manner in capsaicin-sensitive TG neurons. Eugenol also inhibited the VGSC current and AP. These effects were reversed through wash-out. The combination of eugenol and QX-314 was evaluated in the same manner. The combination completely inhibited the VGSC current and AP. However, these effects were not reversed and were continuously blocked even after wash-out. Taken together, our results suggest that, in contrast to the effect of eugenol alone, the combination of eugenol and QX-314 irreversibly and selectively blocked VGSCs in TG neurons expressing TRPV1. Full article
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