scholarly journals Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulations

2019 ◽  
Author(s):  
T. Barbot ◽  
V. Beswick ◽  
C. Montigny ◽  
E. Quiniou ◽  
N. Jamin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Transition ◽  
Phosphorylation Site ◽  
Normal Modes ◽  
General Mechanism ◽  
Regulatory Peptide ◽  
Mechanism Of Inhibition ◽  
Normal Modes Analysis ◽  
The Impact

AbstractSERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well understood. To decipher this mechanism, we have performed normal modes analysis in the all-atom model, with the SERCA1a-SLN complex or the isolated SERCA1a embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304 and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+-deprived E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN, although the SERCA1a-SLN complex was crystallized in an E1-like state. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight in the conformational transition between the E2 and E1 states.Statement of SignificanceThe role of sarco/endoplasmic reticulum calcium ATPase in muscle relaxation is essential. Impairment of its function may result in either cardiac diseases, or myopathies, and also thermogenesis defects. Inhibition of the ATPase by regulatory peptide such as sarcolipin remains unclear. The structure of the ATPase in complex with this peptide was studied by all-atom normal modes analysis, an in silico technique which allows us to decipher the mechanism of inhibition of calcium transport by sarcolipin at a molecular level. Our results open the way to understanding the impact of in vivo misregulation of the ATPase activity by sarcolipin. Development of tools enhancing or preventing interaction between the ATPase and its regulatory peptide could be considered as new therapeutic approaches.

scholarly journals Deciphering the Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations

2021 ◽  
Vol 7 ◽  
Author(s):  
Thomas Barbot ◽  
Veronica Beswick ◽  
Cédric Montigny ◽  
Éric Quiniou ◽  
Nadège Jamin ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Normal Mode Analysis ◽  
Transmembrane Helix ◽  
Phosphorylation Site ◽  
Regulatory Peptides ◽  
Mode Analysis ◽  
General Mechanism ◽  
Atp Binding Site ◽  
Mechanism Of Inhibition ◽  
Comparison Of The Results

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well-understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304, and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

scholarly journals Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)

2004 ◽  
Vol 383 (2) ◽  
pp. 361-370 ◽  
Author(s):  
Elena S. DREMINA ◽  
Victor S. SHAROV ◽  
Keshava KUMAR ◽  
Asma ZAIDI ◽  
Elias K. MICHAELIS ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Conformational Transition ◽  
Direct Interaction ◽  
Molar Ratio ◽  
Endoplasmic Reticulum Calcium ◽  
Oxidative Inactivation ◽  
Complete Inactivation

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2Δ21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2Δ21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2Δ21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2Δ21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2Δ21 to SERCA. In contrast, Bcl-2Δ21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2Δ21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2Δ21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

scholarly journals Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1

2017 ◽  
Vol 37 (16) ◽  
Author(s):  
Michael C. Armstrong ◽  
Sergej Šestak ◽  
Ahmed A. Ali ◽  
Hanan A. M. Sagini ◽  
Max Brown ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphorylation Site ◽  
Spectrometric Analysis ◽  
Activation Loop ◽  
Unfolded Proteins ◽  
Potential Phosphorylation Site ◽  
Content Type ◽  
Mrna Induction ◽  
Better Than

ABSTRACT The bifunctional protein kinase-endoribonuclease Ire1 initiates splicing of the mRNA for the transcription factor Hac1 when unfolded proteins accumulate in the endoplasmic reticulum. Activation of Saccharomyces cerevisiae Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in Escherichia coli identified S837 as another potential phosphorylation site in vivo. Mutation of all five potential phosphorylation sites in the activation loop decreased, but did not completely abolish, splicing of HAC1 mRNA, induction of KAR2 and PDI1 mRNAs, and expression of a β-galactosidase reporter activated by Hac1i. Phosphorylation site mutants survive low levels of endoplasmic reticulum stress better than IRE1 deletions strains. In vivo clustering and inactivation of Ire1 are not affected by phosphorylation site mutants. Mutation of D836 to alanine in the activation loop of phosphorylation site mutants nearly completely abolished HAC1 splicing, induction of KAR2, PDI1, and β-galactosidase reporters, and survival of ER stress, but it had no effect on clustering of Ire1. By itself, the D836A mutation does not confer a phenotype. These data argue that D836 can partially substitute for activation loop phosphorylation in activation of the endoribonuclease domain of Ire1.

scholarly journals Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells

Nutrients ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 2179
Author(s):  
Quentin Escoula ◽  
Sandrine Bellenger ◽  
Michel Narce ◽  
Jérôme Bellenger
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Palmitic Acid ◽  
Er Stress ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Goblet Cells ◽  
The Impact

Diets high in saturated fatty acids (FA) represent a risk factor for the development of obesity and associated metabolic disorders, partly through their impact on the epithelial cell barrier integrity. We hypothesized that unsaturated FA could alleviate saturated FA-induced endoplasmic reticulum (ER) stress occurring in intestinal secretory goblet cells, and consequently the reduced synthesis and secretion of mucins that form the protective mucus barrier. To investigate this hypothesis, we treated well-differentiated human colonic LS174T goblet cells with palmitic acid (PAL)—the most commonly used inducer of lipotoxicity in in vitro systems—or n-9, n-6, or n-3 unsaturated fatty acids alone or in co-treatment with PAL, and measured the impact of such treatments on ER stress and Muc2 production. Our results showed that only eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids protect goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, whereas neither linolenic acid nor n-9 and n-6 FA are able to provide such protection. We conclude that EPA and DHA could represent potential therapeutic nutrients against the detrimental lipotoxicity of saturated fatty acids, associated with type 2 diabetes and obesity or inflammatory bowel disease. These in vitro data remain to be explored in vivo in a context of dietary obesity.

scholarly journals Mdivi-1 Protects CD4+ T Cells against Apoptosis via Balancing Mitochondrial Fusion-Fission and Preventing the Induction of Endoplasmic Reticulum Stress in Sepsis

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
You Wu ◽  
Yong-Ming Yao ◽  
He-Liang Ke ◽  
Lan Ying ◽  
Yao Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Protective Effect ◽  
Mitochondrial Fusion ◽  
Mitochondrial Morphology ◽  
Experimental Sepsis ◽  
The Impact

Apoptosis of CD4+ T cells plays a central role in the progression of sepsis because it is associated with subsequent immunosuppression and the lack of specific treatment. Thus, developing therapeutic strategies to attenuate the apoptosis of CD4+ T cells in sepsis is critical. Several studies have demonstrated that Mdivi-1, which is a selective inhibitor of the dynamin-related protein 1 (Drp1), attenuates apoptosis of myocardial cells and neurons during various pathologic states. The present study revealed the impact of Mdivi-1 on the apoptosis of CD4+ T cells in sepsis and the potential underlying mechanisms. We used lipopolysaccharide (LPS) stimulation and cecal ligation and puncture (CLP) surgery as sepsis models in vitro and in vivo, respectively. Our results showed that Mdivi-1 attenuated the apoptosis of CD4+ T cells both in vitro and in vivo. The potential mechanism underlying the protective effect of Mdivi-1 involved Mdivi-1 reestablishing mitochondrial fusion-fission balance in sepsis, as reflected by the expression of the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) , Drp1 translocation, and mitochondrial morphology, as observed by electron microscopy. Moreover, Mdivi-1 treatment reduced reactive oxygen species (ROS) production and prevented the induction of endoplasmic reticulum stress (ERS) and associated apoptosis. After using tunicamycin to activate ER stress, the protective effect of Mdivi-1 on CD4+ T cells was reversed. Our results suggested that Mdivi-1 ameliorated apoptosis in CD4+ T cells by reestablishing mitochondrial fusion-fission balance and preventing the induction of endoplasmic reticulum stress in experimental sepsis.

scholarly journals A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

2020 ◽  
Author(s):  
Daniele Cultrone ◽  
W. Nathan Zammit ◽  
Eleanor Self ◽  
Benno Postert ◽  
Jeremy ZR Han ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Cancer Progression ◽  
Sequence Data ◽  
Phosphorylation Site ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Tnfaip3 Gene ◽  
Coding Variants ◽  
The Impact

SUMMARYGermline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~400 non-synonymous coding variants which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilises the new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. Similar to A20-deficient mice, A20-deficient zebrafish are hyper-responsive to inflammatory triggers and exhibit spontaneous early lethality. While ectopic addition of human A20 rescued A20-null zebrafish from lethality, missense mutations at two conserved A20 residues, S381A and C243Y reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a C243Y mutation that associates with human autoimmune disease. These data reveal an evolutionarily conserved role for A20, but also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant TNFAIP3 gene variants. This approach could be utilised to investigate genetic variation for other conserved genes.

scholarly journals Myeloma Derived Extracellular Vesicles Containing Endoplasmic Reticulum Remodel the Bone Marrow Microenvironment Towards a Pro-Tumoral Senescent Phenotype

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 689-689
Author(s):  
Yu Sun ◽  
Jayna J Mistry ◽  
Jamie A Moore ◽  
Charlotte Hellmich ◽  
Christopher R Marlein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endoplasmic Reticulum ◽  
Extracellular Vesicles ◽  
Research Funding ◽  
Image Cytometry ◽  
Conditioned Media ◽  
Nsg Mice ◽  
Senescent Phenotype ◽  
The Impact

Multiple myeloma (MM) is a tumor characterised by the generation of large quantities of immunoglobulin which undergoes protein folding and secretion through the endoplasmic reticulum (ER). Many studies have shown that primary MM cells have high ER stress (Lee et al, 2003 PNAS, Obeng et al 2006 Blood and Mimura et al, 2012 Blood,). Moreover, it has also been shown that serum from MM contains significantly more extracellular vesicles (EVs) than serum from normal patients (Caivano et al, 2015 Tumour Biol). Here we explore if these two observations are connected and determine whether primary MM cells export ER inside EVs and the impact this has on the tumor microenvironment. Primary MM and primary MM bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. To determine if MM secrete ER inside EV, patient derived MM and MM cell lines were transduced with rLV.EF.mCherry-ER lentivirus which fluorescently tags the ER. High-resolution imaging combined with image cytometry shows that CD38+ vesicles containing ER are formed by budding from the MM cell surface. Analysis of MM derived EVs using a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that MM actively export ER in the large EVs, approximately 0.6micron-1.5micron in diameter. To track the recipient cell for large EV packaged ER in vivo, U266 MM cell line (transduced with rLV.EF.mCherry-ER9 lentivirus) was injected into the tail of NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence, as evidence of MM derived ER uptake. Murine CD45-/CD31-Ter119-/CD105+/CD140a+ BMSC had increased mCherry fluorescence but not F4/80+/GR1-/CD115int BM macrophages or CD45-/Ter119-/CD31+ endothelial cells or CD45+ leukocytes. Two proteins detected at high levels in the proteomics analysis of large EV were endoplasmic reticulum oxidoreductin 1 (ERO1) and protein disulfide-isomerase (P4B4) which functionally catalyze the formation, breakage and rearrangement of disulfide bonds resulting in the production of superoxide. Next, we analysed large EVs containing ER for superoxide. MM derived ER+ and ER- large EVs were isolated by sorting for mCherry fluorescence (MM transduced with rLV.EF.mCherry-ER9 lentivirus) and Amplex Red assay confirmed that ER+ large EVs had increased levels of H2O2. In vivo analysis of the BMSC from MM engrafted NSG mice confirmed high oxidative stress as measured by increased H2DCFDA fluorescence. To determine the impact of MM derived ER containing EVs on the function of the BMSC isolated ER containing EVs were incubated with BMSC repeatedly for up to 7 days and senescent markers were assessed. Beta-galactosidase staining, p16ink4a gene expression and a senescence associated secretary phenotype (SASP) were all upregulated in BMSC cultured with MM derived ER+ large EV and not ER- large EV. To determine if MM induced BMSC senescence in vivo we injected U266 and primary MM into NSG mice, humanised NSG mice were used as a control. Post MM engraftment, animals were sacrificed and BMSC were isolated by cell sorting for CD45-/CD31-Ter119-/CD105+/CD140a+ cells and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BMSC from U266 and primary MM engrafted NSG mice and not from humanised NSG mice. Knockdown of p16 in BMSC prevents ER+ large EV from inducing a SASP and conditioned media had no effect on MM proliferation compared to conditioned media from ER+ large EV treated BMSC. Finally, we used an NSG mouse model whereby we transplanted p16ink4a KD BMSC or control KD BMSC with MM cells subcutaneously into the flank. MM combined with p16ink4a KD BMSC has reduced tumor volume compared with animals with control KD BMSC. Data indicates that MM secrete ER in large EV and that MM derived EVs containing ER function as a signal which then changes the physiology of BMSC, towards a senescent phenotype which in turn promotes malignant plasma cell survival and proliferation. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

scholarly journals Exosomal ERp44 derived from ER-stressed cells strengthens cisplatin resistance of nasopharyngeal carcinoma

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tian Xia ◽  
Hui Tian ◽  
Kaiwen Zhang ◽  
Siyu Zhang ◽  
Wenhui Chen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nasopharyngeal Carcinoma ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Cisplatin Resistance ◽  
Flow Cytometric Analysis ◽  
Platinum Drug ◽  
Platinum Based Chemotherapy ◽  
The Impact

Abstract Background Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in head and neck. Platinum-based chemotherapy is an important treatment for NPC. However, the molecular mechanism of resistance to platinum drug remains unknown. Endoplasmic reticulum resident protein 44(ERp44), an unfolded protein response (UPR)-induced endoplasmic reticulum(ER) protein, is induced during ER stress. This research explored the mechanism of ERp44 in strengthening cisplatin resistance in NPC. Methods Western blot and immunohistochemistry were used to investigate the expression of ERp44 and Glucose-Regulated Protein 78(GRP78) in NPC. We took CCK8 to detect the role of ERp44 on cell chemosensitivity. Flow cytometric analysis and western blot were taken to analyze cell apoptosis. We performed differential centrifugation to isolate exosomes from serum or conditioned media of cells and analyzed the impact of exosomal ERp44 on cells cisplatin sensitivity. Finally, the results were confirmed in vivo. Results We found the increased expression of ERp44 and GRP78 in NPC and ERp44 was highly expressed in ER-stressed tissues. Cell proliferation was inhibited after cisplatin treatment when ERp44 was knocked down and ERp44 strengthened cisplatin resistance by influencing cell apoptosis and pyroptosis. Then we also collected exosomes and cell viability was increased after the addition of NPC-derived-exosomes with cisplatin treatment. More importantly, our results showed under ERS, NPC cells secreted exosomes containing ERp44 and could transfer them to adjacent cells to strengthen chemoresistance. Conclusion Our data suggested that exosomal ERp44 derived from ER-stressed NPC cells took an inevitable role in NPC chemoresistance and might act as a treatment target.

scholarly journals Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

2022 ◽  
Vol 18 (1) ◽  
pp. e1010159
Author(s):  
Talita B. Gagliardi ◽  
Monty E. Goldstein ◽  
Daniel Song ◽  
Kelsey M. Gray ◽  
Jae W. Jung ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Airway Epithelium ◽  
High Ratio ◽  
Cytopathic Effects ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
Rhinovirus C ◽  
The Impact

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.

scholarly journals A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Daniele Cultrone ◽  
Nathan W. Zammit ◽  
Eleanor Self ◽  
Benno Postert ◽  
Jeremy Z. R. Han ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Cancer Progression ◽  
Sequence Data ◽  
Phosphorylation Site ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Human Sequence ◽  
Coding Variants ◽  
The Impact

Abstract Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.

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