scholarly journals An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin domain and the human cytokine interleukin 6 is efficiently cleaved in planta

2013 ◽  
Vol 12 (3) ◽  
pp. 311-319
Author(s):  
Nausch Henrik ◽  
Mikschofsky Heike ◽  
Koslowski Roswitha ◽  
Steinmann Alain ◽  
Meyer Udo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Interleukin 6 ◽  
In Planta ◽  
Human Cytokine

scholarly journals Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein

2013 ◽  
Vol 12 (2) ◽  
pp. 1987-1995
Author(s):  
Y. Yi ◽  
Y.-Y. Nian ◽  
H.-W. Ji ◽  
H. Zhang ◽  
L. Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Fusion Protein ◽  
Interleukin 6 ◽  
Giant Panda

scholarly journals N-terminal acetylation of the yeast Derlin Der1 is essential for Hrd1 ubiquitin-ligase activity toward luminal ER substrates

2013 ◽  
Vol 24 (7) ◽  
pp. 890-900 ◽  
Author(s):  
Dimitrios Zattas ◽  
David J. Adle ◽  
Eric M. Rubenstein ◽  
Mark Hochstrasser
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Fusion Protein ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Recent Analysis ◽  
Specific Class ◽  
Wild Type ◽  
Ubiquitin Ligase Activity

Two conserved ubiquitin ligases, Hrd1 and Doa10, mediate most endoplasmic reticulum–associated protein degradation (ERAD) in yeast. Degradation signals (degrons) recognized by these ubiquitin ligases remain poorly characterized. Doa10 recognizes the Deg1 degron from the MATα2 transcription factor. We previously found that deletion of the gene (NAT3) encoding the catalytic subunit of the NatB N-terminal acetyltransferase weakly stabilized a Deg1-fusion protein. By contrast, a recent analysis of several MATα2 derivatives suggested that N-terminal acetylation of these proteins by NatB was crucial for recognition by Doa10. We now analyze endogenous MATα2 degradation in cells lacking NatB and observe minimal perturbation relative to wild-type cells. However, NatB mutation strongly impairs degradation of ER-luminal Hrd1 substrates. This unexpected defect derives from a failure of Der1, a Hrd1 complex subunit, to be N-terminally acetylated in NatB mutant yeast. We retargeted Der1 to another acetyltransferase to show that it is the only ERAD factor requiring N-terminal acetylation. Preventing Der1 acetylation stimulates its proteolysis via the Hrd1 pathway, at least partially accounting for the ERAD defect observed in the absence of NatB. These results reveal an important role for N-terminal acetylation in controlling Hrd1 ligase activity toward a specific class of ERAD substrates.

scholarly journals Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Qian Li ◽  
Qiwang Xiang ◽  
Daming Chen ◽  
John Nicholas
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Interleukin 6 ◽  
Kaposi's Sarcoma ◽  
Castleman’S Disease ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Signal Transducer ◽  
Herpesvirus 8 ◽  
Productive Replication

ABSTRACT Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and localized largely to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 proinflammatory and/or angiogenic viral proteins, in HHV-8-associated Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD), in addition to an MCD-related disorder involving systemic elevation of proinflammatory cytokines, including vIL-6 and human IL-6 (hIL-6). In these diseases, lytic (productive) replication, in addition to viral latency, is believed to play a critical role. Proreplication activity of vIL-6 has been identified experimentally in PEL and endothelial cells, but the relative contributions of different vIL-6 interactions have not been established. Productive interactions of vIL-6 with the IL-6 signal transducer, gp130, can occur within the ER, but vIL-6 also interacts in the ER with a nonsignaling receptor called vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2), calnexin, and VKORC1v2- and calnexin-associated proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII). Here, we report the systematic characterization of interaction-altered vIL-6 variants and the lytic phenotypes of recombinant viruses expressing selected variants. Our data identify the critical importance of vIL-6 and its ER-localized activity via gp130 to productive replication in inducible SLK (epithelial) cells, absence of detectable involvement of vIL-6 interactions with VKORC1v2, GlucII, or UGGT1, and the insufficiency and lack of direct contributory effects of extracellular signaling by vIL-6 or hIL-6. These findings, obtained through genetics-based approaches, complement and extend previous analyses of vIL-6 activity. IMPORTANCE Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) was the first viral IL-6 homologue to be identified. Experimental and clinical evidence suggests that vIL-6 is important for the onset and/or progression of HHV-8-associated endothelial-cell and B-cell pathologies, including AIDS-associated Kaposi’s sarcoma and multicentric Castleman’s disease. The protein is unusual in its poor secretion from cells and its intracellular activity; it interacts, directly or indirectly, with a number of proteins beyond the IL-6 signal transducer, gp130, and can mediate activities through these interactions in the endoplasmic reticulum. Here, we report the characterization with respect to protein interactions and signal-transducing activity of a panel of vIL-6 variants and utilization of HHV-8 mutant viruses expressing selected variants in phenotypic analyses. Our findings establish the importance of vIL-6 in HHV-8 productive replication and the contributions of individual vIL-6-protein interactions to HHV-8 lytic biology. This work furthers understanding of the biological significance of vIL-6 and its unique intracellular interactions.

Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker

2003 ◽  
Vol 96 (1) ◽  
pp. 38-46 ◽  
Author(s):  
Kiyoshi Yasukawa ◽  
Shigeo Tsuchiya ◽  
Teiji Ekida ◽  
Hiroshi Iida ◽  
Teruhiko Ide ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Interleukin 6 ◽  
Interleukin 6 Receptor

scholarly journals Promotion of Endoplasmic Reticulum-Associated Degradation of Procathepsin D by Human Herpesvirus 8-Encoded Viral Interleukin-6

2015 ◽  
Vol 89 (15) ◽  
pp. 7979-7990 ◽  
Author(s):  
Daming Chen ◽  
John Nicholas
Keyword(s):  
Endoplasmic Reticulum ◽  
Interleukin 6 ◽  
Cathepsin D ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Gp130 Receptor ◽  
Epoxide Reductase ◽  
Productive Replication

ABSTRACTThe interleukin-6 homologue (viral interleukin-6 [vIL-6]) of human herpesvirus 8 is implicated in viral pathogenesis due to its proproliferative, inflammatory, and angiogenic properties, effected through gp130 receptor signaling. In primary effusion lymphoma (PEL) cells, vIL-6 is expressed latently and is essential for normal cell growth and viability. This is mediated partly via suppression of proapoptotic cathepsin D (CatD) via cocomplexing of the endoplasmic reticulum (ER)-localized CatD precursor, pro-CatD (pCatD), and vIL-6 with the previously uncharacterized ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). vIL-6 suppression of CatD occurs also during reactivated productive replication in PEL cells and is likely to contribute to proreplication functions of vIL-6. Here, we report that vIL-6 suppresses CatD through vIL-6, VKORC1v2, and pCatD association with components of the ER-associated degradation (ERAD) machinery. In transfected cells, expression of vIL-6 along with CatD led to proteasome-dependent (inhibitor-sensitive) decreases in CatD levels and the promotion of pCatD polyubiquitination. Depletion of particular ERAD-associated isomerases, lectins, and translocon components, including ERAD E3 ubiquitin ligase HRD1, diminished suppression of CatD by vIL-6. Coprecipitation assays identified direct or indirect interactions of VKORC1v2, vIL-6, and pCatD with translocon proteins (SEL1L and/or HRD1) and ERAD-associated lectins OS9 and XTP3-B. Endogenous CatD expression in PEL cells was increased by depletion of ERAD components, and suppression of CatD by vIL-6 overexpression in PEL cells was dependent on HRD1. Our data reveal a new mechanism of ER-localized vIL-6 activity and further characterize VKORC1v2 function.IMPORTANCEHuman herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6), unlike cellular IL-6 proteins, is secreted inefficiently and sequestered mainly in the endoplasmic reticulum (ER), from where it can signal through the gp130 receptor. We have recently reported that vIL-6 also associates with a novel membrane protein termed vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2) and mediates suppression of VKORC1v2-cointeracting cathepsin D, a stress-released proapoptotic protein negatively impacting HHV-8 latently infected primary effusion lymphoma (PEL) cell viability and reactivated virus productive replication. Here, we have examined the mechanistic basis of the VKORC1v2–vIL-6 interaction-dependent suppression of cathepsin D and have found that this novel activity of vIL-6 is mediated through coassociation of VKORC1v2, procathepsin D, and vIL-6 with components of the ER-associated degradation (ERAD) machinery. Our findings provide information of significance for potential antiviral and therapeutic targeting of VKORC1v2-mediated vIL-6 activities and also indicate the nature of VKORC1v2 function in normal cell biology.

Studies of cytokinin receptor–phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum

2018 ◽  
Vol 45 (2) ◽  
pp. 192 ◽  
Author(s):  
Sergey N. Lomin ◽  
Yulia A. Myakushina ◽  
Dmitry V. Arkhipov ◽  
Olga G. Leonova ◽  
Vladimir I. Popenko ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Endoplasmic Reticulum ◽  
Active Form ◽  
Kinase Assay ◽  
Phosphoryl Group ◽  
Subcellular Localisation ◽  
In Planta ◽  
Cytokinin Receptors ◽  
Receptor Dimers

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

Prevention of neuron and oligodendrocyte degeneration by interleukin-6 (IL-6) and IL-6 receptor/IL-6 fusion protein in organotypic hippocampal slices

2004 ◽  
Vol 25 (2) ◽  
pp. 301-311 ◽  
Author(s):  
Marina Pizzi ◽  
Ilenia Sarnico ◽  
Flora Boroni ◽  
Marina Benarese ◽  
Michel Dreano ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Interleukin 6 ◽  
Hippocampal Slices

scholarly journals A Dual Target-Directed Single Domain-Based Fusion Protein Against Interleukin-6 Receptor Decelerate Experimental Arthritis Progression Via Modulating JNK Expression

2021 ◽  
Author(s):  
Xiaole Chen ◽  
Yize Bian ◽  
Yongqing Xie ◽  
Ningning Zheng ◽  
Kaimei Nie ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Interleukin 6 ◽  
Expression System ◽  
Single Domain ◽  
No Production ◽  
Dependent Manner ◽  
Drug Study ◽  
Prokaryotic Expression System ◽  
Interleukin 6 Receptor ◽  
Dual Target

Abstract The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signalling that cause collateral damage to protective signalling cascades. The single domain chain firstly discovered in Camelidae displays fully functional ability in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. In this study, we established a simple prokaryotic expression system for a dual target-directed single domain-based Fusion Protein against the interleukin-6 receptor and human serum, albumin, the recombinant anti-IL-6R fusion protein (VHH-0031). VHH-0031 exhibited potent anti-inflammatory effects produced by LPS on cell RAW264.7, where the major cytokines and NO production were down-regulated after 24 hr incubation with VHH-0031 in a dose-dependent manner. In vivo, VHH-0031 presented significant effects on the degree reduction of joint swelling in the adjuvant-induced arthritis (AIA) rat, having a healthier appearance compared to the dexamethasone. The expression level of JNK protein in the VHH-0031 group was significantly decreased, demonstrating that VHH-0031 provides a low-cost and desirable effect in the treatment of more widely patients.

scholarly journals Mitochondrial fusion protein 2 regulates endoplasmic reticulum stress in preeclampsia

2021 ◽  
Vol 22 (2) ◽  
pp. 165-170
Author(s):  
Dandan Sun ◽  
Hui Zhu ◽  
Ling Ai ◽  
Hanbing Wu ◽  
Yanting Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Fusion Protein ◽  
Mitochondrial Fusion
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