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Search results for: Nterm

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#32611767   2020/07/01 To Up

Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.

Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNA  Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influence the Nterm conformation. To explore the latter, we used Sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm-ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with ssRNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm-ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak, but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.
Sheng Liu, Maryanne Refaei, Shuohui Liu, Aaron Decker, Jennifer M Hinerman, Andrew B Herr, Mike Howell, Karin Musier-Forsyth, Pearl Tsang

2152 related Products with: Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.

100ul 100ul100ug1000 TESTS/0.65ml100ug1 ml96 assays 100ul500 mg4 Membranes/Box

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#32332915   2020/04/24 To Up

HSPA12A attenuates lipopolysaccharide-induced liver injury through inhibiting caspase-11-mediated hepatocyte pyroptosis via PGC-1α-dependent acyloxyacyl hydrolase expression.

Liver dysfunction is strongly associated with poor survival of sepsis patients. Cytosolic lipopolysaccharide (LPS) sensing by Caspase-4/5/11 for pyroptosis activation is a major driver of the development of sepsis. Studies in macrophages and endothelial cells have demonstrated that LPS is inactivated by acyloxyacyl hydrolase (AOAH) and leading to desensitizing Caspase-4/5/11 to LPS. However, little is known about the cytosolic LPS-induced pyroptosis in hepatocytes during sepsis. Heat shock protein 12A (HSPA12A) is a novel member of the HSP70 family. Here, we report that LPS increased HSPA12A nuclear translocation in hepatocytes, while knockout of HSPA12A (Hspa12a) in mice promoted LPS-induced acute liver injury. We also noticed that the LPS-induced Caspase-11 activation and its cleavage of gasdermin D (GSDMD) to produce the membrane pore-forming GSDMD (markers of pyroptosis) were greater in livers of Hspa12a mice compared with its wild type controls. Loss- and gain-of-function studies showed that HSPA12A deficiency promoted, whereas HSPA12A overexpression inhibited, cytosolic LPS accumulation, Caspase-11 activation and GSDMD generation in primary hepatocytes following LPS incubation. Notably, LPS-induced AOAH expression was suppressed by HSPA12A deficiency, whereas AOAH overexpression reversed the HSPA12A deficiency-induced promotion of LPS-evoked and Caspase-11-mediated pyroptosis of hepatocytes. In-depth molecular analysis showed that HSPA12A interacted directly with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and increased its nuclear translocation, thereby inducing AOAH expression for cytosolic LPS inactivation, which ultimately leading to inhibition of the Caspase-11 mediated pyroptosis of hepatocytes. Taken together, these findings revealed HSPA12A as a novel player against LPS-induced liver injury by inhibiting cytosolic LPS-induced hepatocyte pyroptosis via PGC-1α-mediated AOAH expression. Therefore, targeting hepatocyte HSPA12A represents a viable strategy for the management of liver injury in sepsis patients.
Jiali Liu, Shuya Du, Qiuyue Kong, Xiaojin Zhang, Surong Jiang, Xiaofei Cao, Yuehua Li, Chuanfu Li, Huaqun Chen, Zhengnian Ding, Li Liu

1920 related Products with: HSPA12A attenuates lipopolysaccharide-induced liver injury through inhibiting caspase-11-mediated hepatocyte pyroptosis via PGC-1α-dependent acyloxyacyl hydrolase expression.

N/A N/A 200 assays100 ug400 assays10μg/vial20 100 200 0.1mg

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#31596312   2019/10/07 To Up

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry.

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.
Elizangela Farias, Fhabiane Bezerra, Djane Clarys Baia-da-Silva, Yury Oliveira Chaves, Tatiana Bacry Cardoza, Maria Edilene Martins de Almeida, Lucas Barbosa Oliveira, Pritesh Lalwani, Patrícia Puccinelli Orlandi, Marcus Vinicius Guimaraes Lacerda, Stefanie Costa Pinto Lopes, Paulo Afonso Nogueira

2747 related Products with: A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry.

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#31152664   // To Up

Spatial Assessment of Contact Between Humans and Anopheles and Aedes Mosquitoes in a Medium-Sized African Urban Setting, Using Salivary Antibody-Based Biomarkers.

Anarchic and poorly controlled urbanization led to an increased risk of mosquito-borne diseases (MBD) in many African cities. Here, we evaluate the spatial heterogeneity of human exposure to malaria and arboviral disease vectors in an urban area of northern Senegal, using antibody-based biomarkers of exposure to Anopheles and Aedes mosquito bites.
André B Sagna, Daouda Kassié, Agnès Couvray, Akré Maurice Adja, Emmanuel Hermann, Gilles Riveau, Gérard Salem, Florence Fournet, Franck Remoue

1808 related Products with: Spatial Assessment of Contact Between Humans and Anopheles and Aedes Mosquitoes in a Medium-Sized African Urban Setting, Using Salivary Antibody-Based Biomarkers.

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#31036506   2019/04/26 To Up

MicroRNA-135b alleviates MPP-mediated Parkinson's disease in in vitro model through suppressing FoxO1-induced NLRP3 inflammasome and pyroptosis.

The present study focused on the novel roles and the underlying mechanisms of miR-135b in pyroptosis of MPP-induced Parkinson's disease (PD). We established an in vitro PD model induced by MPP. Our results demonstrated miR-135b was lower while FoxO1 was inversely higher in MPP-treated SH-SY5Y and PC-12 cells. Luciferase reporter assay showed FoxO1 was a downstream target of miR-135b. MiR-135b mimics suppressed MPP-induced pyroptosis and the upregulation of TXNIP, NLRP3, Caspase-1, ASC, GSDMD and IL-1β. Moreover, FoxO1 overexpression had no effect on miR-135b but reversed its own downregulation caused by miR-135b mimics. Meanwhile, overexpression of FoxO1 abolished the inhibitory effects of miR-135b on pyroptosis and reversed the downregulation of pyroptotic genes and LDH release. In summary, miR-135b played a protective role in Parkinson's disease via inhibiting pyroptosis by targeting FoxO1. MiR-135b might serve as a potential therapeutic target in the treatment of Parkinson's disease.
Rong Zeng, Di-Xian Luo, Hai-Peng Li, Qi-Shan Zhang, Sheng-Suo Lei, Ji-Hua Chen

1021 related Products with: MicroRNA-135b alleviates MPP-mediated Parkinson's disease in in vitro model through suppressing FoxO1-induced NLRP3 inflammasome and pyroptosis.

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#29941881   // To Up

Author Correction: N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

This Article contains a URL for a publically available whole-genome browser ( http://nterm.listeriomics.pasteur.fr ). However, due to technical constraint, this website has been replaced with an alternative ( https://listeriomics.pasteur.fr ).
Francis Impens, Nathalie Rolhion, Lilliana Radoshevich, Christophe Bécavin, Mélodie Duval, Jeffrey Mellin, Francisco García Del Portillo, M Graciela Pucciarelli, Allison H Williams, Pascale Cossart

1286 related Products with: Author Correction: N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes.

100μg100μg4 Membranes/Box96 assays96 samples4 Membranes/Box20 µl (10 mM)4 Membranes/Box100 assays4 Membranes/Box100 tests100 assays

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#29898893   2018/06/13 To Up

Mechanism of membrane pore formation by human gasdermin-D.

Gasdermin-D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N-terminal domain (GSDMD) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMD insertion, oligomerization, and pore formation are poorly understood. Here, we apply high-resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMD inserts and assembles in membranes. We observe GSDMD inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMD assembles arc-, slit-, and ring-shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5. Using time-lapse AFM, we monitor how GSDMD assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. Our observations translate into a mechanistic model of GSDMD transmembrane pore assembly, which is likely shared within the gasdermin protein family.
Estefania Mulvihill, Lorenzo Sborgi, Stefania A Mari, Moritz Pfreundschuh, Sebastian Hiller, Daniel J Müller

1581 related Products with: Mechanism of membrane pore formation by human gasdermin-D.

4 Membranes/Box4 Membranes/Box4 Membranes/Box4 Membranes/Box4 Membranes/Box0.1 mg4 Membranes/Box4 Membranes/Box 100ul4 Membranes/Box5pk4 Membranes/Box

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#29212938   2018/02/12 To Up

Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase.

The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22. Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.
Ju-Bei Yen, Ling-Huei Wei, Lee-Wen Chen, Li-Yu Chen, Chien-Hui Hung, Shie-Shan Wang, Pey-Jium Chang

1120 related Products with: Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase.

10 mg2.5 mg100 mg 25 G5 mg 5 G1 g100ug Lyophilized 5 G1 mg (4000 assays)5 x 50 Pieces/case

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#27906987   2016/12/01 To Up

Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area.

Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes.
Emmanuel Elanga Ndille, Souleymane Doucoure, Anne Poinsignon, François Mouchet, Sylvie Cornelie, Eric D'Ortenzio, Jean Sébastien DeHecq, Franck Remoue

2346 related Products with: Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area.

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#27833005   2016/11/08 To Up

Comparative analysis of South African norovirus GII.4 strains identifies minor recombinant variants.

Recombination within the norovirus (NoV) GII.4 genotype is well documented as a mechanism by which novel variants evolve. Norovirus GII.4 has been the predominant NoV genotype detected in South Africa (SA) in recent years and putative NoV recombinants were previously identified in SA based on partial regions of the viral genome. The objective of this study was to determine the complete genome sequence of representative NoV GII.4 variants that have circulated in SA between 2009 and 2013 and to compare major and minor GII.4 variants based on nucleotide sequence. The complete genomes of 11/27 GII.4 strains could be amplified in three or five overlapping segments, these included major variants New_Orleans_2009 and Sydney_2012 as well as three types of minor GII.4 variants. Phylogenetic and recombination analysis identified GII.4 recombinants with breakpoints located at or near the ORF1/2 junction. Apart from recombinants already recognised as major variants (GII.P4 New_Orleans_2009/GII.4 Sydney_2012 (n=2) and GII.Pe/GII.4 Sydney_2012 (n=2)) four further recombinant strains were detected (GII.P4 New_Orleans_2009/GII.4 Hunter_2004 (n=1) and GII.P4 Yerseke_2006a/GII.4 Apeldoorn_2007 (n=3)) that were attributed to three distinct minor variants. The encoded proteins with the highest diversity were p48 (Nterm), p22, VP1 and VP2. Analysis of antigenic sites in VP1 revealed mutations at epitopes A, B, C, D and E, with epitopes A and D being most variable. The high variation at epitope D was reflected in structural differences in models of GII.4 variants in the epitope D loop region (aa 393-395). Major and minor variants could not be distinguished based on specific sequence differences. HBGA-binding studies will be necessary to assess the effect of the observed amino acid differences in the P2 domain of these GII.4 strains.
J C Botha, M B Taylor, J Mans

2711 related Products with: Comparative analysis of South African norovirus GII.4 strains identifies minor recombinant variants.

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