αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.

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Mitochondrial LonP1 protease is implicated in the degradation of unstable Parkinson's disease-associated DJ-1/PARK 7 missense mutants

Scientific Reports ◽

10.1038/s41598-021-86847-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Raúl Sánchez-Lanzas ◽

José G. Castaño

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mitochondrial Pathway ◽

Mitochondrial Matrix ◽

Wild Type ◽

Strong Reduction ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Biochemical Fractionation ◽

Early Onset Parkinson’S Disease

AbstractDJ-1/PARK7 mutations are linked with familial forms of early-onset Parkinson's disease (PD). We have studied the degradation of untagged DJ-1 wild type (WT) and missense mutants in mouse embryonic fibroblasts obtained from DJ-1-null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants L10P, M26I, A107P, P158Δ, L166P, E163K, and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E, and A179T are as stable as DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158Δ, L166P, E163K, and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of the mitochondrial-associated DJ-1 mutants A107P, P158Δ, L166P, E163K, and L172Q but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis.

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Plasma Membrane Estrogen Receptors Exist and Functions as Dimers

Molecular Endocrinology ◽

10.1210/me.2004-0115 ◽

2004 ◽

Vol 18 (12) ◽

pp. 2854-2865 ◽

Cited By ~ 218

Author(s):

Mahnaz Razandi ◽

Ali Pedram ◽

Istvan Merchenthaler ◽

Geoffrey L. Greene ◽

Ellis R. Levin

Keyword(s):

Breast Cancer ◽

Endothelial Cells ◽

Plasma Membrane ◽

Estrogen Receptors ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Membrane Receptors ◽

Cell Physiology ◽

Wild Type ◽

Double Knockout

Abstract A small pool of estrogen receptors (ERα and -β) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17β-estradiol (E2). Interestingly, in endothelial cells made from ERα /ERβ homozygous double-knockout mice, membrane ERα or ERβ are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERα. Only wild-type ERα supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsα and Gqα at the membrane in cells expressing the wild-type, but not the dimer mutant, ERα. Intact, but not dimer mutant, ERα also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERα. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.

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The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Communications Biology ◽

10.1038/s42003-021-01963-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Anurag Kumar Sinha ◽

Kristoffer Skovbo Winther

Keyword(s):

Active Sites ◽

Molecular Switch ◽

Cell Physiology ◽

Synthetase Activity ◽

Wild Type ◽

Functional Studies ◽

Loop Region ◽

A Site ◽

Trna Binding

AbstractBacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

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Absence of the αvβ3 Integrin Dictates the Time-Course of Angiogenesis in the Hypoxic Central Nervous System: Accelerated Endothelial Proliferation Correlates with Compensatory Increases in α5β1 Integrin Expression

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.276 ◽

2010 ◽

Vol 30 (5) ◽

pp. 1031-1043 ◽

Cited By ~ 27

Author(s):

Longxuan Li ◽

Jennifer V Welser ◽

Richard Milner

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Time Course ◽

Αvβ3 Integrin ◽

Integrin Expression ◽

Brain Endothelial Cells ◽

Endothelial Proliferation ◽

Β3 Integrin ◽

Α5β1 Integrin

Cerebral angiogenesis is an important adaptive response to hypoxia. As the αvβ3 integrin is induced on angiogenic vessels in the ischemic central nervous system (CNS), and the suggested angiogenic role for this integrin in other systems, it is important to determine whether the αvβ3 integrin is an important mediator of cerebral angiogenesis. αvβ3 integrin expression was examined in a model of cerebral hypoxia, in which mice were subject to hypoxia (8% O2) for 0, 4, 7, or 14 days. Immunofluorescence and western blot analysis revealed that in the hypoxic CNS, αvβ3 integrin was strongly induced on angiogenic brain endothelial cells (BEC), along with its ligand vitronectin. In the hypoxia model, β3 integrin-null mice showed no obvious defect in cerebral angiogenesis. However, early in the angiogenic process, BEC in these mice showed an increased mitotic index that correlated closely with increased α5 integrin expression. In vitro experiments confirmed α5 integrin upregulation on β3 integrin-null BEC, which also correlated with increased BEC proliferation on fibronectin. These studies confirm hypoxic induction of αvβ3 integrin on angiogenic vessels, but suggest distinct roles for the BEC integrins αvβ3 and α5β1 in cerebral angiogenesis, with αvβ3 having a nonessential role, and α5β1 promoting BEC proliferation.

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Increased susceptibility of purinergic receptor-deficient mice to lung infection withPseudomonasaeruginosa

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00428.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. L890-L895 ◽

Cited By ~ 24

Author(s):

Cara Geary ◽

Henry Akinbi ◽

Tom Korfhagen ◽

Jean-Etienne Fabre ◽

Richard Boucher ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Purinergic Receptors ◽

Tissue Injury ◽

Purinergic Receptor ◽

Intratracheal Instillation ◽

Lavage Fluid ◽

Sublethal Dose ◽

Wild Type ◽

Double Knockout

Purinergic receptors are expressed throughout the respiratory system in diverse cell types. The efficiency of mucus clearance in the airways, the cascade leading to tissue injury, and inflammation are modulated by autocrine/paracrine release of nucleotides and signaling by purinergic receptors. We assessed the role of purinergic receptors in innate host defense of the lung in vivo by infecting mice deficient in P2Y1, P2Y2, or both receptors with intratracheal instillation of Pseudomonas aeruginosa. After P. aeruginosa challenge, all double knockout (P2Y1/P2Y2−/−) mice succumbed within 30 h of challenge, whereas 85% of the wild-type mice survived. Thirty-three percent of wild-type mice survived beyond 96 h. Single knockout mice, P2Y1−/−, or P2Y2−/−, exhibited intermediate survivals. Twenty-four hours following intratracheal instillation of a sublethal dose of P. aeruginosa, the level of total protein in bronchoalveolar lavage fluid was 1.8-fold higher in double knockout than in wild-type mice ( P < 0.04). Total cell count in bronchoalveolar lavage fluids at 4 h and levels of IL-6 and macrophage inflammatory protein-2 in lung homogenates at 24 h postchallenge were significantly reduced in P2Y1/P2Y2−/− mice relative to wild-type mice. These findings suggest that purinergic receptors exert a protective role against infection of the lungs by P. aeruginosa by decreasing protein leak and enhancing proinflammatory cytokine response.

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S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux

Cells ◽

10.3390/cells10040929 ◽

2021 ◽

Vol 10 (4) ◽

pp. 929

Author(s):

Aleksandra Hać ◽

Karolina Pierzynowska ◽

Anna Herman-Antosiewicz

Keyword(s):

Degradation Process ◽

Underlying Mechanism ◽

Stress Conditions ◽

Autophagic Flux ◽

Ratiometric Fluorescence ◽

Mouse Embryonic Fibroblasts ◽

Double Knockout ◽

Embryonic Fibroblasts ◽

Microtubule Network ◽

Tubulin Acetylation

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

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The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish

Blood ◽

10.1182/blood-2010-11-321182 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4978-4987 ◽

Cited By ~ 40

Author(s):

Elzbieta Pluskota ◽

James J. Dowling ◽

Natalie Gordon ◽

Jeffrey A. Golden ◽

Dorota Szpak ◽

...

Keyword(s):

Endothelial Cells ◽

Critical Role ◽

Integrin Αvβ3 ◽

Tube Formation ◽

Basement Membranes ◽

Cytoskeletal Protein ◽

Integrin Expression ◽

Partial Reduction ◽

Wild Type ◽

Developmental Defects

Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2+/− mice. RM1 prostate tumors grown in kindlin-2+/− mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2+/− mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2+/− mice. Vessels in the kindlin-2+/− mice were leaky, and BM transplantation from kindlin-2+/− to WT mice did not correct this defect. Endothelial cells derived from kindlin-2+/− mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

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Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6303 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6303-6310 ◽

Cited By ~ 37

Author(s):

L Yu ◽

M A Gorovsky

Keyword(s):

Protein Sequence ◽

Tetrahymena Thermophila ◽

Histone H3 ◽

Constitutive Expression ◽

Histone Variants ◽

Wild Type ◽

Ciliated Protozoan ◽

Knockout Mutant ◽

Double Knockout ◽

Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

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Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

Eukaryotic Cell ◽

10.1128/ec.00255-08 ◽

2008 ◽

Vol 8 (2) ◽

pp. 217-229 ◽

Cited By ~ 36

Author(s):

Vellaisamy Ramamoorthy ◽

Edgar B. Cahoon ◽

Mercy Thokala ◽

Jagdeep Kaur ◽

Jia Li ◽

...

Keyword(s):

Fusarium Graminearum ◽

Type Strain ◽

Wild Type Strain ◽

Fungal Growth ◽

Wild Type ◽

Knockout Mutant ◽

Double Knockout ◽

Disease Symptoms ◽

Plant Defensins ◽

Antifungal Action

ABSTRACT The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

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Structural and Nonstructural Genes Contribute to the Genetic Diversity of RNA Viruses

mBio ◽

10.1128/mbio.01871-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 11

Author(s):

Natalie D. Collins ◽

Andrew S. Beck ◽

Steven G. Widen ◽

Thomas G. Wood ◽

Stephen Higgs ◽

...

Keyword(s):

Genetic Diversity ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Rna Viruses ◽

Rna Virus ◽

Infectious Clone ◽

Vaccine Virus ◽

Wild Type ◽

Rna Dependent Rna Polymerase ◽

Replication Complex

ABSTRACT One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity. IMPORTANCE With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.

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