scholarly journals HSC70 Inhibits Spring Viremia of Carp Virus Replication by Inducing MARCH8-Mediated Lysosomal Degradation of G Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Chen Li ◽  
Lin Shi ◽  
Yan Gao ◽  
Yuanan Lu ◽  
Jing Ye ◽  
...  
Keyword(s):  
G Protein ◽  
Defense Mechanism ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Lysosomal Degradation ◽  
Spectrometry Analysis ◽  
Glycoprotein G ◽  
Multicomponent Complex ◽  
Host Defense Mechanism ◽  
Novel Host

As a fierce pathogen, spring viremia of carp virus (SVCV) can cause high mortality in the common carp, and its glycoprotein (G protein) is a component of the viral structure on the surface of virion, which is crucial in viral life cycle. This report adopted tandem affinity purification (TAP), mass spectrometry analysis (LC-MS/MS), immunoprecipitation, and confocal microscopy assays to identify Heat shock cognate protein 70 (HSC70) as an interaction partner of SVCV G protein. It was found that HSC70 overexpression dramatically inhibited SVCV replication, whereas its loss of functions elicited opposing effects on SVCV replication. Mechanistic studies indicate that HSC70 induces lysosomal degradation of ubiquitinated-SVCV G protein. This study further demonstrates that Membrane-associated RING-CH 8 (MARCH8), an E3 ubiquitin ligase, is critical for SVCV G protein ubiquitylation and leads to its lysosomal degradation. Furthermore, the MARCH8 mediated ubiquitylation of SVCV G protein required the participation of HSC70 through forming a multicomponent complex. Taken together, these results demonstrate that HSC70 serves as a scaffold for MARCH8 and SVCV G, which leads to the ubiquitylation and degradation of SVCV G protein and thus inhibits viral replication. These findings have established a novel host defense mechanism against SVCV.

scholarly journals Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

2020 ◽  
Author(s):  
Bo Wei ◽  
Patrick Willems ◽  
Jingjing Huang ◽  
Caiping Tian ◽  
Jing Yang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Technological Advancement ◽  
Amino Acid Residues ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Mixed Disulfide ◽  
And Function

ABSTRACTIn proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1–like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

scholarly journals Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

2019 ◽  
Vol 116 (38) ◽  
pp. 19136-19144 ◽  
Author(s):  
Giel P. Göertz ◽  
Joyce W. M. van Bree ◽  
Anwar Hiralal ◽  
Bas M. Fernhout ◽  
Carmen Steffens ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Zika Virus ◽  
Affinity Purification ◽  
Mechanistic Model ◽  
Virus Transmission ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Small Interfering Rnas ◽  
Spectrometry Analysis ◽  
Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

scholarly journals Interaction between PHB2 and Enterovirus A71 VP1 Induces Autophagy and Affects EV-A71 Infection

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 414 ◽  
Author(s):  
Weitao Su ◽  
Shan Huang ◽  
Huimin Zhu ◽  
Bao Zhang ◽  
Xianbo Wu
Keyword(s):  
Defense Mechanism ◽  
Foot And Mouth Disease ◽  
Underlying Mechanism ◽  
Host Protein ◽  
Mass Spectrometry Analysis ◽  
Structural Protein ◽  
Viral Protein Synthesis ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Enterovirus A71

Enterovirus A71 (EV-A71) is a major pathogen that causes severe and fatal cases of hand-foot-and-mouth disease (HFMD). HFMD caused by EV-A71 seriously endangers children’s health. Although autophagy is an important antiviral defense mechanism, some viruses have evolved strategies to utilize autophagy to promote self-replication. EV-A71 can utilize autophagy vesicles as replication scaffolds, indicating that EV-A71 infection is closely related to its autophagy induction mechanism. VP1, a structural protein of EV-A71, has been reported to induce autophagy, but the underlying mechanism is still unclear. In this study, we found that the C-terminus (aa 251–297) of VP1 induces autophagy. Mass spectrometry analysis suggested that prohibitin 2 (PHB2) interacts with the C-terminus of the EV-A71 VP1 protein, and this was further verified by coimmunoprecipitation assays. After PHB2 knockdown, EV-A71 replication, viral particle release, and viral protein synthesis were reduced, and autophagy was inhibited. The results suggest that PHB2 interaction with VP1 is essential for induction of autophagy and the infectivity of EV-A71. Furthermore, we confirmed that EV-A71 induced complete autophagy that required autolysosomal acidification, thus affecting EV-A71 infection. In summary, this study revealed that the host protein PHB2 is involved in an autophagy mechanism during EV-A71 infection.

scholarly journals ProbingN-glycoprotein microheterogeneity by lectin affinity purification-mass spectrometry analysis

2019 ◽  
Vol 10 (19) ◽  
pp. 5146-5155 ◽  
Author(s):  
Di Wu ◽  
Jingwen Li ◽  
Weston B. Struwe ◽  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Protein Level ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Spectrometry Analysis ◽  
Lectin Affinity ◽  
Affinity Purification Mass Spectrometry

A lectin affinity purification-mass spectrometry approach to characterize lectin-reactive glycoproteoforms and elucidate lectin specificities at the intact protein level.

scholarly journals Identification of Novel Surface Proteins of Anaplasma phagocytophilum by Affinity Purification and Proteomics

2007 ◽  
Vol 189 (21) ◽  
pp. 7819-7828 ◽  
Author(s):  
Yan Ge ◽  
Yasuko Rikihisa
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Affinity Purification ◽  
The United States ◽  
Capillary Liquid Chromatography ◽  
Etiologic Agent ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

scholarly journals Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration

2014 ◽  
Vol 25 (5) ◽  
pp. 658-668 ◽  
Author(s):  
Pascale Daou ◽  
Salma Hasan ◽  
Dennis Breitsprecher ◽  
Emilie Baudelet ◽  
Luc Camoin ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Affinity Purification ◽  
Cortical Microtubule ◽  
Leading Edge ◽  
Large Family ◽  
Mass Spectrometry Analysis ◽  
Cell Cortex ◽  
Spectrometry Analysis ◽  
Microtubule Capture

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.

scholarly journals Hsp110 is required for spindle length control

2012 ◽  
Vol 198 (4) ◽  
pp. 623-636 ◽  
Author(s):  
Taras Makhnevych ◽  
Philip Wong ◽  
Oxana Pogoutse ◽  
Franco J. Vizeacoumar ◽  
Jack F. Greenblatt ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Spindle Assembly ◽  
S Phase ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Exchange ◽  
Spectrometry Analysis ◽  
Nucleotide Exchange Factor ◽  
Spindle Elongation ◽  
Chaperone Complex

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone–protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70–Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.

scholarly journals Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

Diseases ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 64 ◽  
Author(s):  
Ziying Han ◽  
Michael Schwoerer ◽  
Philip Hicks ◽  
Jingjing Liang ◽  
Gordon Ruthel ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Hemorrhagic Fever ◽  
Lassa Fever ◽  
Cellular Protein ◽  
Negative Regulator ◽  
Host Protein ◽  
Ww Domain ◽  
Host Defense Mechanism ◽  
Lassa Fever Virus ◽  
Novel Host

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

scholarly journals Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

2021 ◽  
Author(s):  
Ching-Seng Ang ◽  
Joanna Sacharz ◽  
Michael G Leeming ◽  
Shuai Nie ◽  
Swati Varshney ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Modern Biology ◽  
Spectrometry Analysis ◽  
Affinity Enrichment ◽  
Unbiased Manner ◽  
Gas Phase Ion

Co-immunoprecipitation of proteins coupled to mass spectrometry has transformed modern biology understanding of protein interaction networks. These approaches exploit the selective isolation of tagged proteins by affinity enrichment / purification to identify protein binding partners at scale and in an unbiased manner. In instances where a suitable antibody is not be available it is common to graft synthetic tags such as FLAG or His Tags onto target protein sequences allowing the use of commercially available and validated antibodies for affinity purification. To allow the selective elution of protein complexes competitive displacement using a large molar excess of the tag peptide is widely used. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of a contaminating peptide. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device can be applied to FLAG-Tag and His-Tag pull down assay to increase the depth of protein coverage in these experiments. By excluding tag peptides based on their ion mobility profiles we demonstrate that single compensation voltage, or stepped compensation voltages strategies can significantly increase the coverage of total proteins by up to 2.5-fold and unique proteins by up to 15-fold versus experiments that do not use FAIMS. Combined these results highlight FAIMS is able to improve proteome depth by excluding interfering peptides without the need for additional sample handling or altering sample preparation protocols.

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