scholarly journals Surface-Exposed Proteins of Ehrlichia chaffeensis

2007 ◽  
Vol 75 (8) ◽  
pp. 3833-3841 ◽  
Author(s):  
Yan Ge ◽  
Yasuko Rikihisa
Keyword(s):  
Vaccine Development ◽  
Affinity Purification ◽  
Hypothetical Protein ◽  
Capillary Liquid Chromatography ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Ehrlichia Chaffeensis ◽  
Monocytic Leukemia ◽  
Spectrometry Analysis ◽  
Host Interactions

ABSTRACT The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.

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 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.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

2021 ◽  
Vol 14 (680) ◽  
pp. eaaw4673
Author(s):  
Natalia Zamorano Cuervo ◽  
Audray Fortin ◽  
Elise Caron ◽  
Stéfany Chartier ◽  
Nathalie Grandvaux
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Host Interactions ◽  
In Cells

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

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 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 Targeting the endothelial progenitor cell surface proteome to identify novel mechanisms that mediate angiogenic efficacy in a rodent model of vascular disease

2013 ◽  
Vol 45 (21) ◽  
pp. 999-1011 ◽  
Author(s):  
Catherine C. Kaczorowski ◽  
Timothy J. Stodola ◽  
Brian R. Hoffmann ◽  
Anthony R. Prisco ◽  
Pengyuan Y. Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Vascular Disease ◽  
Rodent Model ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Major Advance ◽  
Endothelial Progenitor ◽  
Angiogenic Potential ◽  
Spectrometry Analysis

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials suggest autologous EPC-based therapy may be effective in treatment of vascular diseases. Albeit promising, variability in the efficacy of EPCs associated with underlying disease states has hindered the realization of EPC-based therapy. Here we first identify and characterize EPC dysfunction in a rodent model of vascular disease (SS/Mcwi rat) that exhibits impaired angiogenesis. To identify molecular candidates that mediate the angiogenic potential of these cells, we performed a broad analysis of cell surface protein expression using chemical labeling combined with mass spectrometry. Analysis revealed EPCs derived from SS/Mcwi rats express significantly more type 2 low-affinity immunoglobulin Fc-gamma (FCGR2) and natural killer 2B4 (CD244) receptors compared with controls. Genome-wide sequencing (RNA-seq) and qt-PCR confirmed isoforms of CD244 and FCGR2a transcripts were increased in SS/Mcwi EPCs. EPCs with elevated expression of FCGR2a and CD244 receptors are predicted to increase the probability of SS/Mcwi EPCs being targeted for death, providing a mechanistic explanation for their reduced angiogenic efficacy in vivo. Pathway analysis supported this contention, as “key” molecules annotated to cell death paths were differentially expressed in the SS/Mcwi EPCs. We speculate that screening and neutralization of cell surface proteins that “tag” and impair EPC function may provide an alternative approach to utilizing incompetent EPCs in greater numbers, as circulating EPCs are depleted in patients with vascular disease. Overall, novel methods to identify putative targets for repair of EPCs using discovery-based technologies will likely provide a major advance in the field of regenerative medicine.

scholarly journals Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

2002 ◽  
Vol 70 (1) ◽  
pp. 368-379 ◽  
Author(s):  
Silvia Montigiani ◽  
Fabiana Falugi ◽  
Maria Scarselli ◽  
Oretta Finco ◽  
Roberto Petracca ◽  
...  
Keyword(s):  
Chlamydia Pneumoniae ◽  
Genetic Manipulation ◽  
Respiratory Infections ◽  
Surface Protein ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Definition Of

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

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.

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