scholarly journals SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253089
Author(s):  
Christine Vazquez ◽  
Sydnie E. Swanson ◽  
Seble G. Negatu ◽  
Mark Dittmar ◽  
Jesse Miller ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Mrna Translation ◽  
Viral Proteins ◽  
Interferon Signaling ◽  
Protein Levels ◽  
Global Pandemic ◽  
Activation Pathways ◽  
Irf3 Activation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

scholarly journals PBRM1 Cooperates with YTHDF2 to Control HIF-1α Protein Translation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1425
Author(s):  
Alena Shmakova ◽  
Mark Frost ◽  
Michael Batie ◽  
Niall S. Kenneth ◽  
Sonia Rocha
Keyword(s):  
Protein Expression ◽  
Cellular Response ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Protein Translation ◽  
Signalling Pathways ◽  
Independent Function ◽  
Protein Levels ◽  
Associated Proteins ◽  
Core Components

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.

scholarly journals In Silico Exploration of Repurposing and Optimizing Traditional Chinese Medicine Rutin for Possibly Inhibiting SARS-CoV-2's Main Protease

2020 ◽  
Author(s):  
Tien Huynh ◽  
Haoran Wang ◽  
Wendy Cornell ◽  
Binquan Luan
Keyword(s):  
In Silico ◽  
Molecular Mechanisms ◽  
Herbal Medicines ◽  
Viral Proteins ◽  
Biological Functions ◽  
Rna Dependent Rna Polymerase ◽  
Global Pandemic ◽  
Main Protease ◽  
Dynamics Simulations ◽  
Viral Target

<div>Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic with very limited specific treatments. To fight COVID-19, various traditional antiviral medicines haveb been prescribed in China to infected patients with mild to moderate symptoms and received unexpected success in controlling the disease. However, the molecular mechanisms of how these herbal medicines interact with the virus have remained elusive. It is well known that the main protease (Mpro) of SARS-CoV-2 plays an important role in maturation of many viral proteins such as the RNA-dependent RNA polymerase. Here,we explore the underlying molecular mechanisms of the computationally determined top candidate–rutin, a key component in many traditional antiviral medicines such as Lianhuaqinwen and Shuanghuanlian, for inhibiting the viral target–Mpro. Using in silico methods (docking and molecular dynamics simulations), we revealed the dynamics and energetics of rutin when interacting with the Mpro of SARS-CoV-2, suggesting that the highly hydrophilic rutin molecule can be bound inside the Mpro’ pocket (active site) and possibly inhibit its biological functions. In addition, we optimized the structure of rutin and designed a more hydrophobic analog which satisfies the rule of five for western medicines and demonstrated that it possesses a much stronger binding affinity to the SARS-COV-2’s Mpro.<br></div>

scholarly journals GRN, NOTCH3, FN1, and PINK1 expression in eutopic endometrium – potential biomarkers in the detection of endometriosis – a pilot study

2020 ◽  
Vol 37 (11) ◽  
pp. 2723-2732
Author(s):  
Isabell Holzer ◽  
Amanda Machado Weber ◽  
Anne Marshall ◽  
Alexander Freis ◽  
Julia Jauckus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laparoscopic Surgery ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Reproductive Age ◽  
Tissue Samples ◽  
Protein Levels ◽  
Clinical Biomarkers ◽  
Mrna Gene ◽  
Severity Of The Disease

Abstract Purpose Endometriosis (EM) is a common gynecological disease affecting 10–15% of women of reproductive age. However, molecular mechanisms and pathogenesis are still not completely understood. Furthermore, due to the absence of a reliable clinical biomarker, the only viable method for the often-delayed definitive diagnosis is laparoscopic surgery. Our objective was to analyze molecular differences of selected endometrial proteins and genes of women suffering from different stages of EM compared with healthy women to evaluate potential clinical biomarkers. Methods We analyzed eutopic endometrial tissue samples from women undergoing a laparoscopic surgery (n = 58). mRNA gene expression of progranulin (GRN), neurogenic locus notch homolog protein (NOTCH3), fibronectin (FN1), and PTEN-induced kinase 1 (PINK1) was analyzed using qRT-PCR. Protein expression was determined using ELISA and immunohistochemistry. Results Significant differences in gene expression between the different stages of the disease were noted for GRN, NOTCH3, FN1, and PINK1 (p < 0.05). The endometrium of women with minimal EM (ASRM I) showed the highest mRNA expression. Protein levels of GRN and FN1 on the other hand were significantly decreased in the endometrium of women with EM compared with those of healthy controls. Furthermore, for GRN and FN1, we could detect a correlation of protein expression with the severity of the disease. Conclusion Our findings suggest a potential use of GRN and FN1 as clinical biomarkers to detect endometriosis. In addition, GRN, NOTCH3, FN1, and PINK1 could potentially be useful to differentiate between the underlying stages of the disease. However, a validation with a larger study population is needed.

scholarly journals PBRM1 Cooperates with YTHDF2 to Control HIF-1Alpha Protein Translation

2021 ◽  
Author(s):  
Alena Shmakova ◽  
Mark Frost ◽  
Niall S Kenneth ◽  
Sonia Rocha
Keyword(s):  
Protein Expression ◽  
Cellular Response ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Protein Translation ◽  
Signalling Pathways ◽  
Independent Function ◽  
Protein Levels ◽  
Associated Proteins ◽  
Core Components

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here we investigated how PBRM1 controls HIF-1alpha activity. We find that PBRM1 is required for HIF-1alpha transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1alpha mRNA translation, as absence of PBRM1 results in reduced activly transalting HIF-1alpha mRNA. Interestingly, we find that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1alpha mRNA is m6A modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1alpha mRNA and reduction of YTHDF2 results in reduced HIF-1alpha protein expression in cells. Our results identify a SWI/SNF independent function for PBRM1, interacting with HIF-1alpha mRNA and the epitranscriptome machinery. Furthermore, our results suggests that the epitranscriptome associated proteins play a role in the control of hypoxia signalling pathways

MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation

2012 ◽  
Vol 302 (12) ◽  
pp. F1630-F1639 ◽  
Author(s):  
S. A. Ásgeirsdóttir ◽  
C. van Solingen ◽  
N. F. Kurniati ◽  
P. J. Zwiers ◽  
P. Heeringa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Molecular Control ◽  
Protein Levels ◽  
Tnf Α ◽  
Inflammatory Stimuli ◽  
Endothelial Cell Adhesion Molecules ◽  
Differential Responsiveness

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291–299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

l-Mimosine blocks cell proliferation via upregulation of B-cell translocation gene 2 and N-myc downstream regulated gene 1 in prostate carcinoma cells

2012 ◽  
Vol 302 (4) ◽  
pp. C676-C685 ◽  
Author(s):  
Li-Chuan Chung ◽  
Ke-Hung Tsui ◽  
Tsui-Hsia Feng ◽  
Shiow-Ling Lee ◽  
Phei-Lang Chang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
B Cell ◽  
Prostate Carcinoma ◽  
Molecular Mechanisms ◽  
Carcinoma Cells ◽  
Transcriptional Level ◽  
Lncap Cells ◽  
Protein Levels

l-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 ( Btg2) regulates the G1/S transition phases of the cell cycle. N- myc downstream regulated gene 1 ( Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of l-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of l-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. l-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and l-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. l-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that l-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or l-mimosine in LNCaP cells. Our results indicated that hypoxia and l-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. l-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells.

scholarly journals Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress

Hypertension ◽  
2019 ◽  
Vol 73 (1) ◽  
pp. 179-189 ◽  
Author(s):  
Hong Li ◽  
Qiang Li ◽  
Yixuan Zhang ◽  
Wenting Liu ◽  
Bo Gu ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Dihydrofolate Reductase ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Therapeutic Option ◽  
Protein Levels ◽  
Treatment Of Hypertension ◽  
Mrna And Protein Expression

We have shown that hydrogen peroxide (H 2 O 2 ) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H 2 O 2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H 2 O 2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H 2 O 2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H 2 O 2 on DHFR promoter activity. H 2 O 2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H 2 O 2 . E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)–infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H 2 O 2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.

scholarly journals Interleukin-12-mediated expression of matrix metalloproteinases in human periodontal ligament fibroblasts involves in NF-κB activation

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Li Miao ◽  
Shujun Zhan ◽  
Jiyan Liu
Keyword(s):  
Matrix Metalloproteinases ◽  
Protein Expression ◽  
Periodontal Ligament ◽  
Nuclear Translocation ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Interleukin 12 ◽  
Periodontal Ligament Fibroblasts ◽  
Protein Levels ◽  
Mrna And Protein Expression ◽  
Human Periodontal Ligament Fibroblasts

Interleukin-12 (IL-12) is a proinflammatory cytokine, and its increased level correlates with the severity of periodontitis. However, its role in the pathogenesis of tooth periapical lesions is controversial and has not been completely clarified. The present study aimed to investigate whether IL-12 affects the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human periodontal ligament fibroblasts (hPDLFs). After treatment with IL-12 for different times, real-time PCR and Western blotting were used to determine the mRNA and protein levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2, respectively. ELISA was applied to measure MMPs and TIMPs secretion production. The results indicated that IL-12 significantly increased the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13, but down-regulated MMP-2 and MMP-9 mRNA and protein expression in the hPDLFs. Furthermore, IL-12 (10 ng/ml) enhanced the secreted protein production of MMP-1, MMP-3, and MMP-13, and conversely lowered MMP-2 and MMP-9 secretion levels. However, IL-12 treatment did not exert a significant effect on the mRNA and protein levels of TIMP-1 and TIMP-2 and their secreted production. Additionally, IL-12 increased the phosphorylated levels of IκBα and nuclear factor-κB P65 (NF-κB P65), and promoted NF-κB P65 subunit nuclear translocation. Pretreatment with NF-κB inhibitor not only attenuated IL-12-induced IκBα and NF-κB P65 phosphorylation and inhibited NF-κB P65 subunit into nucleus, but also antagonized IL-12-mediated MMP-1, MMP-2, MMP-3, MMP-9, and MMP-13 expression in the hPDLFs. These findings indicate that NF-κB-dependent activation is possibly indispensable for IL-12-mediated MMP expression in hPDLFs.

scholarly journals In Silico Exploration of Repurposing and Optimizing Traditional Chinese Medicine Rutin for Possibly Inhibiting SARS-CoV-2's Main Protease

2020 ◽  
Author(s):  
Tien Huynh ◽  
Haoran Wang ◽  
Wendy Cornell ◽  
Binquan Luan
Keyword(s):  
In Silico ◽  
Molecular Mechanisms ◽  
Herbal Medicines ◽  
Viral Proteins ◽  
Biological Functions ◽  
Rna Dependent Rna Polymerase ◽  
Global Pandemic ◽  
Main Protease ◽  
Dynamics Simulations ◽  
Viral Target

<div>Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic with very limited specific treatments. To fight COVID-19, various traditional antiviral medicines haveb been prescribed in China to infected patients with mild to moderate symptoms and received unexpected success in controlling the disease. However, the molecular mechanisms of how these herbal medicines interact with the virus have remained elusive. It is well known that the main protease (Mpro) of SARS-CoV-2 plays an important role in maturation of many viral proteins such as the RNA-dependent RNA polymerase. Here,we explore the underlying molecular mechanisms of the computationally determined top candidate–rutin, a key component in many traditional antiviral medicines such as Lianhuaqinwen and Shuanghuanlian, for inhibiting the viral target–Mpro. Using in silico methods (docking and molecular dynamics simulations), we revealed the dynamics and energetics of rutin when interacting with the Mpro of SARS-CoV-2, suggesting that the highly hydrophilic rutin molecule can be bound inside the Mpro’ pocket (active site) and possibly inhibit its biological functions. In addition, we optimized the structure of rutin and designed a more hydrophobic analog which satisfies the rule of five for western medicines and demonstrated that it possesses a much stronger binding affinity to the SARS-COV-2’s Mpro.<br></div>

scholarly journals Secreted Wnt6 mediates diabetes-associated centrosome amplification via its receptor FZD4

2020 ◽  
Vol 318 (1) ◽  
pp. C48-C62 ◽  
Author(s):  
Qin Ju He ◽  
Pu Wang ◽  
Qin Qin Liu ◽  
Qi Gui Wu ◽  
Yuan Fei Li ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Cell Culture ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Integration Site ◽  
Cellular Protein ◽  
Centrosome Amplification ◽  
Protein Levels

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of β-catenin. The treatment also activated β-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or β-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-β-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of β-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

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