scholarly journals Expression Levels of Glycoprotein O (gO) Vary between Strains of Human Cytomegalovirus, Influencing the Assembly of gH/gL Complexes and Virion Infectivity

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Le Zhang ◽  
Momei Zhou ◽  
Richard Stanton ◽  
Jeremy Kamil ◽  
Brent J. Ryckman
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Purifying Selection ◽  
Selective Advantage ◽  
Immune Recognition ◽  
Expression Levels

ABSTRACT The tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the endoplasmic reticulum (ER). This assembly process clearly differs among strains, since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much lower levels of gH/gL/UL128-131 than ME. Remaining questions include (i) what are the mechanisms behind these assembly differences, and (ii) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) open reading frame (ORF) differs in 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced the infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression levels were within 4-fold between TR and ME, but the gO expression level was 20-fold lower for ME, which suggested differences in mRNA transcription, translation, or rapid ER-associated degradation of gO. trans-Complementation of gO expression during ME replication gave a 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in the assembly of gH/gL complexes result from differences in the expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than those for higher gO expression. IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo. This may have important implications for virus-host interactions, such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

scholarly journals Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity

2018 ◽  
Author(s):  
Le Zhang ◽  
Momei Zhou ◽  
Richard Stanton ◽  
Jeremy Kamil ◽  
Brent J. Ryckman
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Purifying Selection ◽  
Selective Advantage ◽  
Immune Recognition ◽  
Virion Assembly

ABSTRACTTropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the mechanisms behind these assembly differences, and 2) do differences reflectin vitroculture adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL complexes result from differences in expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than for higher gO expression.IMPORTANCESpecific genetic differences between independently isolated HCMV strains may result from purifying selection onde novomutations arising during propagation in culture, or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability presentin vivo. This may have important implications for virus-host interactions such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

scholarly journals 905 FASN prevents immunogenicity of irradiated glioblastoma by inhibiting ER stress

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A950-A950
Author(s):  
Mara De Martino ◽  
Camille Daviaud ◽  
Claire Vanpouille-Box
Keyword(s):  
Er Stress ◽  
Lipid Accumulation ◽  
Fatty Acid Synthase ◽  
Immune Escape ◽  
De Novo ◽  
Lipid Synthesis ◽  
Adult Brain ◽  
Immune Recognition

BackgroundGlioblastoma (GBM) is the most aggressive and incurable adult brain tumor. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD) subsequent to endoplasmic reticulum (ER) stress. However, RT also exacerbates potent immunosuppressive mechanisms that facilitate immune evasion. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is emerging as a mechanism of therapy resistance and immune escape. Here, we hypothesize that RT induces FASN to promote GBM survival and evade immune recognition by inhibiting ER stress and ICD.MethodsTo determine if lipid synthesis is altered in response to RT, we first assessed FASN expression by western blot (WB) and lipid accumulation by BODIPY staining in murine (CT2A and GL261) and human (U118) GBM cell lines. Next, FASN expression was blocked in CT2A cells using CRISPR-Cas9 or an inducible shRNA directed against Fasn to evaluate ICD and ER stress markers by ELISA, WB, and electron microscopy. Finally, CT2AshFASN cells or its non-silencing control (CT2AshNS) were orthotopically implanted and FASN knockdown was induced by feeding the mice with doxycycline. The immune contexture was determined by in situ immunofluorescence (n=3/group). Remaining mice were followed for survival (n=7/group).ResultsWe found that in vitro irradiation of GBM cells induces lipid accumulation in a dose-dependent fashion; an effect that is magnified over time lasting at least 6/7 days. Consistent with these findings, FASN expression was upregulated in irradiated GBM cells. Confirming the role of FASN, RT-induced accumulation of lipids was reverted when GBM cells were incubated with a FASN inhibitor. Next, we found that FASN ablation in CT2A cells induces mitochondria disruption and was sufficient to increase the expression of the ER stress makers BIP and CHOP. Along similar lines, shFASN enhances the secretion of the ICD markers HMGB1, IFN-beta and CXCL10 in irradiated CT2A cells. In vivo, CT2AshFASN tumors presented increased infiltration of CD11c+ cells and CD8+ T cells, consistent with prolonged mice survival (56 days vs. 28 days for CT2AshNS). Importantly, 43% of CT2AshFASN-bearing mice remained tumor-free for more than 70 days, while none of the CT2AshNS-bearing mice survived.ConclusionsAltogether, our data suggest that FASN-mediated lipid synthesis is an important mechanism to prevent ER stress, ICD, and anti-tumor immune responses in GBM. While much work remains to be done, our data propose FASN as a novel therapeutic target to overcome immunosuppression and sensitize GBM to immunotherapies.

scholarly journals Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 165 ◽  
Author(s):  
Andrew J. Hayes ◽  
Jiulia Satiaputra ◽  
Louise M. Sternicki ◽  
Ashleigh S. Paparella ◽  
Zikai Feng ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Resistance Rate ◽  
Biotin Protein Ligase ◽  
Essential Enzyme

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

scholarly journals HIF-1–dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia

2005 ◽  
Vol 202 (11) ◽  
pp. 1493-1505 ◽  
Author(s):  
Holger K. Eltzschig ◽  
Parween Abdulla ◽  
Edgar Hoffman ◽  
Kathryn E. Hamilton ◽  
Dionne Daniels ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Nucleoside Transporter ◽  
In Vivo Models ◽  
Equilibrative Nucleoside Transporter ◽  
Hypoxia Inducible Factor 1 ◽  
And Function

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

scholarly journals Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

2021 ◽  
Vol 9 ◽  
Author(s):  
Chuanjie Zhang ◽  
Yan Shen ◽  
Lili Gao ◽  
Xiaojing Wang ◽  
Da Huang ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Cancer Genome ◽  
Clinicopathological Parameters ◽  
Expression Levels

ObjectiveThe aim of this study is to investigate the biological functions and the underlying mechanisms of DNA polymerase epsilon subunit 2 (POLE2) in renal cell carcinoma (RCC).MethodsThe datasets of POLE2 expression in The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) and International Cancer Genome Consortium (ICGC) databases was selected and the correlation between POLE2 and various clinicopathological parameters was analyzed. The POLE2 expression in RCC tissues was examined by immunohistochemistry. The POLE2 knockdown cell lines were constructed. In vitro and in vivo experiments were carried out to investigate the function of POLE2 on cellular biology of RCC, including cell viability assay, clone formation assay, flow cytometry, wound-healing assay, Transwell assay, qRT-PCR, Western blot, etc. Besides, microarray, co-immunoprecipitation, rescue experiment, and Western blot were used to investigate the molecular mechanisms underlying the functions of POLE2.ResultsPOLE2 was overexpressed in RCC tissues, and high expression of POLE2 was correlated with poor prognosis of RCC. Furthermore, knockdown of POLE2 significantly inhibited cell proliferation, migration, and facilitated apoptosis in vitro. In vivo experiments revealed that POLE2 attenuated RCC tumorigenesis and tumor growth. we also illuminated that stanniocalcin 1 (STC1) was a downstream gene of POLE2, which promoted the occurrence and development of RCC. Besides, knockdown of POLE2 significantly upregulated the expression levels of Bad and p21 while the expression levels of HSP70, IGF-I, IGF-II, survivin, and sTNF-R1 were significantly downregulated. Western blot analysis also showed that knockdown of POLE2 inhibited the expression levels of Cancer-related pathway proteins including p-Akt, CCND1, MAPK9, and PIK3CA.ConclusionKnockdown of POLE2 attenuates RCC cells proliferation and migration by regulating STC1, suggesting that POLE2-STC1 may become a potential target for RCC therapy.

In vitro reconstitution of autophagic processes

2020 ◽  
Vol 48 (5) ◽  
pp. 2003-2014
Author(s):  
Jahangir Md. Alam ◽  
Nobuo N. Noda
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
The Past ◽  
Atg Proteins ◽  
In Vitro Reconstitution ◽  
Complicated Process

Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence

1998 ◽  
Vol 111 (6) ◽  
pp. 759-767
Author(s):  
G. Herrmann ◽  
P. Brenneisen ◽  
M. Wlaschek ◽  
J. Wenk ◽  
K. Faisst ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Molecular Data ◽  
Cellular Aging ◽  
Premature Aging ◽  
Functional Changes ◽  
Uva Irradiation ◽  
High Level

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of &gt;120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

scholarly journals GABRQ is Overexpressed and Correlated with Tumor Progression in Breast Cancer

2021 ◽  
Author(s):  
Ran Mei ◽  
Xichun Cui ◽  
Lili Zheng ◽  
Li Jingyi
Keyword(s):  
Breast Cancer ◽  
Molecular Mechanisms ◽  
Tumor Development ◽  
Bioinformatic Analysis ◽  
Gamma Aminobutyric Acid ◽  
Nude Mouse Model ◽  
Expression Levels ◽  
The Public

Abstract Background: Breast cancer (BRCA) is the most common type of women's cancer with a high incidence. The function of gamma-aminobutyric acid A receptor θ subunit (GABRQ) has been studied in other cancers. The results demonstrated that the expression levels of GABRQ were closely associated with tumor prognosis. However, the functions and mechanisms of GABRQ in BRCA remain unclear.Materials and methods: We used the public genome datasets and a tissue microarray (TMA) cohort to analyze the GABRQ expression levels. We performed Immunohistochemistry (IHC) and Western blot to determine GABRQ expression in BRCA cell lines and tissues. Cell proliferation was assessed by EDU assay and colony formation assay. Transwell assay was carried out to investigate the cell invasion ability in vitro and Xenograft nude mouse model was constructed to test the function of GABRQ on tumor growth in vivo. Moreover, we utilized bioinformatic analysis to identify the potential molecular mechanisms mediated by GABRQ modification in BRCA.Results: GABRQ was markedly up-regulated in BRCA tissues, and the expression levels of GABRQ were closely associated with BRCA prognosis. Functional analysis elucidated that knockdown of GABRQ could suppress BRCA cell growth and invasion in vitro, and inhibit tumor development in vivo. Moreover, we found that GABRQ overexpression activated the EMT signaling pathway.Conclusions: These results demonstrated that the function of GABRQ in BRCA progression provided potential prognostic predictors for BRCA patients.

scholarly journals The Regulatory RNA ern0160 Confers a Potential Selective Advantage to Enterococcus faecium for Intestinal Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Sophie Reissier ◽  
Killian Le Neindre ◽  
Valérie Bordeau ◽  
Loren Dejoies ◽  
Audrey Le Bot ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Molecular Mechanisms ◽  
Selective Advantage ◽  
Regulatory Rna ◽  
Intestinal Colonization ◽  
Potential Implication ◽  
Significant Difference

The aim of this study was to evaluate the role of the regulatory small RNA (sRNA) Ern0160 in gastrointestinal tract (GIT) colonization by Enterococcus faecium. For this purpose, four strains of E. faecium were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). Strains were studied both in vitro and in vivo, alone and in competitive assays. In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured single while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. Then, in vivo experiments were performed using a GIT colonization mouse model. Several existing models of GIT colonization were compared while a novel one, combining ceftriaxone and amoxicillin, was developed. A GIT colonization was performed with each strain alone, and no significant difference was noticed. By contrast, significant results were obtained with co-colonization experiments. With WT + Δ0160 suspension, a significant advantage for the WT strain was observed from day 5 to the end of the protocol, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_pAT29 suspension, the strain with the empty vector took the advantage from day 3 to the end of the protocol, suggesting a deleterious effect of ern0160 overexpression. Altogether, these findings demonstrate the potential implication of Ern0160 in GIT colonization of E. faecium. Further investigations are needed for the identification of sRNA target(s) in order to decipher underlying molecular mechanisms.

scholarly journals Endothelial Sphingolipid De Novo Synthesis Controls Blood Pressure by Regulating Signal Transduction and NO via Ceramide

Hypertension ◽  
2020 ◽  
Vol 75 (5) ◽  
pp. 1279-1288 ◽  
Author(s):  
Anna Cantalupo ◽  
Linda Sasset ◽  
Antonella Gargiulo ◽  
Luisa Rubinelli ◽  
Ilaria Del Gaudio ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Signal Transduction ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Control Flow ◽  
Serine Palmitoyltransferase ◽  
Blood Pressure Homeostasis

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus–related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)—the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

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