Cellular proteomic analysis of porcine circovirus type 2 and classical swine fever virus coinfection in porcine kidney‐15 cells using isobaric tags for relative and absolute quantitation‐coupled LC‐MS/MS

Viral coinfection or superinfection in host has caused public health concern and huge economic losses of farming industry. The influence of viral coinfection on cellular protein abundance is essential for viral pathogenesis. Based on a coinfection model for porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV) developed previously by our laboratory, isobaric tags for relative and absolute quantitation (iTRAQ)‐coupled LC‐MS/MS proteomic profiling was performed to explore the host cell responses to PCV2‐CSFV coinfection. Totally, 3932 proteins were identified in three independent mass spectrometry analyses. Compared with uninfected cells, 304 proteins increased (fold change >1.2) and 198 decreased (fold change <0.833) their abundance in PCV2‐infected cells (p < 0.05), 60 and 61 were more and less abundant in CSFV‐infected cells, and 196 and 158 were more and less abundant, respectively in cells coinfected with PCV2 and CSFV. Representative differentially abundant proteins were validated by quantitative real‐time PCR, Western blotting and confocal laser scanning microscopy. Bioinformatic analyses confirmed the dominant role of PCV2, and indicated that mitochondrial dysfunction, nuclear factor erythroid 2‐related factor 2 (Nrf2)‐mediated oxidative stress response and apoptosis signaling pathways might be the specifical targets during PCV2‐CSFV coinfection.     

Mass spectrometry based proteomic studies on viruses and hosts--a review

In terms of proteomic research in the 21st century, the realm of virology is still regarded as an enormous challenge mainly brought by three aspects, namely, studying on the complex proteome of the virus with unexpected variations, developing more accurate analytical techniques as well as understanding viral pathogenesis and virus–host interaction dynamics. Progresses in these areas will be helpful to vaccine design and antiviral drugs discovery. Mass spectrometry based proteomics have shown exceptional display of capabilities, not only precisely identifying viral and cellular proteins that are functionally, structurally, and dynamically changed upon virus infection, but also enabling us to detect important pathway proteins. In addition, many isolation and purification techniques and quantitative strategies in conjunction with MS can significantly improve the sensitivity of mass spectrometry for detecting low-abundant proteins, replenishing the stock of virus proteome and enlarging the protein–protein interaction maps. Nevertheless, only a small proportion of the infectious viruses in both of animal and plant have been studied using this approach. As more virus and host genomes are being sequenced, MS-based proteomics is becoming an indispensable tool for virology. In this paper, we provide a brief review of the current technologies and their applications in studying selected viruses and hosts.     

Alpha‐1 antitrypsin variants in plasma from HIV‐infected patients revealed by proteomic and glycoproteomic analysis

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV‐positive patients. Through comparative proteome analysis of normal and HIV‐positive plasma samples, 19 differentially expressed protein spots related to 12 non‐redundant proteins were identified by ESI‐ion trap MS. Among these, the 130‐kDa isoform of α‐1‐antitrypsin was found to be decreased in HIV‐positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS‐2‐D‐PAGE reference gel and protein sequence comparisons of the 40‐kDa protein showed homology with α‐1‐antitrypsin minus the N‐terminus, and its identity was further confirmed by 1‐D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40‐kDa variant of α‐1‐antitrypsin found in the plasma of HIV‐positive individuals may prove to be a potentially useful biomarker for anti‐HIV research according to bioinformatics analysis.     

Mechanical characterization of HIV‐1 with a solid‐state nanopore sensor

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo‐type human immunodeficiency virus type 1 at sub‐micron scale. Non‐infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.     

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small‐molecule inhibitors of DOT1L such as FED1 are potential anti‐cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo‐reactive and “clickable” affinity‐based probes (AfBPs), P1 and P2 , which were cell‐permeable and structural mimics of FED1 . The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub‐cellular localization properties of the probes were subsequently studied in live‐cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1 . Finally with P1 / P2 , large‐scale cell‐based proteome profiling, followed by quantitative LC‐MS/MS, was carried out to identify potential cellular off‐targets of FED1 . Amongst the more than 100 candidate off‐targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off‐target of FED1 by preliminary validation experiments including pull‐down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).     

液相色谱-质谱联用分析流感病毒感染引起宿主蛋白类泛素化修饰

干扰素刺激基因15编码蛋白质(Interferon stimulated gene 15 kDa protein, ISG15)是最早被鉴定的类泛素分子蛋白质,在病毒感染和免疫调节等方面具有重要作用。本研究利用免疫沉淀技术将被类泛素 ISG15修饰的蛋白富集纯化,采用液相色谱-质谱联用技术对流感病毒感染 A549宿主细胞过程中产生的类泛素 ISG15修饰蛋白进行了分析。实验结果表明,在流感病毒感染的实验组 A549细胞中,鉴定到了22种来源于宿主细胞的ISG15修饰的蛋白,包括类泛素蛋白 ISG15、细胞周期蛋白-T1、热休克蛋白71、钙调素结合蛋白、真核翻译起始因子等,以及1种来源于流感病毒的非结构蛋白 NS1。在鉴定的22种宿主蛋白中,有6种蛋白在未感染病毒的对照组 A549细胞中也得到鉴定,包括膜联蛋白 A1、果糖二磷酸醛缩酶 A、线粒体三磷酸腺苷合成酶亚基 g、烯醇化酶、肌动蛋白、微管蛋白。生物信息学分析表明,流感病毒感染引起的 ISG15修饰的宿主蛋白分别归属于9个不同的蛋白分类,包括细胞骨架蛋白、分子伴侣蛋白、酶调节剂、核酸结合蛋白、激酶类、转移酶类、转录因子、氧化还原酶类以及结构蛋白。本研究为大规模分析鉴定 ISG15修饰蛋白提供了一种特异、有效的研究方法。     

A Molecular Chameleon for Mapping Subcellular Polarity in an Unfolded Proteome Environment

Environmental polarity is an important factor that drives biomolecular interactions to regulate cell function. Herein, a general method of using the fluorogenic probe NTPAN‐MI is reported to quantify the subcellular polarity change in response to protein unfolding. NTPAN‐MI fluorescence is selectively activated upon labeling unfolded proteins with exposed thiols, thereby reporting on the extent of proteostasis. NTPAN‐MI also reveals the collapse of the host proteome caused by influenza A virus infection. The emission profile of NTPAN‐MI contains information of the local polarity of the unfolded proteome, which can be resolved through spectral phasor analysis. Under stress conditions that disrupt different checkpoints of protein quality control, distinct patterns of dielectric constant distribution in the cytoplasm can be observed. However, in the nucleus, the unfolded proteome was found to experience a more hydrophilic environment across all the stress conditions, indicating the central role of nucleus in the stress response process.     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Affinity‐Driven Covalent Modulator of the Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) Cascade

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)‐(+)‐isochaihulactone ( 1 ) exhibited significant inhibition against multiple‐drug‐resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off‐target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β‐unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI‐oriented activity‐based proteome profiling. By MS/MS and computer‐guided molecular biology approaches, an affinity‐driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the “ON/OFF” switch of apoptosis through non‐canonically nuclear GAPDH translocation, which bypasses the common apoptosis‐resistant route of MDR cancers.     

Cellular pharmacokinetics and pharmacodynamics mechanisms of ginkgo diterpene lactone and its modulation of P‐glycoprotein expression in human SH‐SY5Y cells

Ginkgo diterpene lactone (GDL) is the raw material for ginkgo diterpene lactone meglumine injection, which is used for treating cerebral ischemia. The aims of this study were to explore the cellular pharmacokinetics of GDL in whole cells and subcellular fractions, and detect cellular pharmacodynamics on the human SH‐SY5Y cells induced by oxygen–glucose deprivation and reoxygenation (OGD/R). Firstly, a simple, sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for assessing the amount of ginkgolide A (GA), B (GB) and K (GK) in cellular/subcellular samples. Then, phosphatidylserine and mitochondria membrane potential were assayed to evaluate the extent of apoptosis effect. The study showed that the cellular/subcellular accumulation of GA and GB were increased in a concentration‐dependent manner; the levels of GA and GB in cytosol were the highest among these subcellular organelles. Meanwhile, GDL also attenuated the OGD/R‐induced increases in the percentage of apoptotic and mitochondria membrane potential. In addition, verapamil increased the rate and amount of GA and GB entering cellular/subcellular compartments through inhibition of P‐glycoprotein activity, and promoted the protective effect of GDL. The present study reports the cellular pharmacokinetics profiles of GA and GB in normal and OGD/R‐induced SH‐SY5Y cells in vitro for the first time, which provided valuable information for clinical safety application.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigative proteomics: identification of an unknown plant virus from infected plants using mass spectrometry

We describe the identification of a previously uncharacterized plant virus that is capable of infecting Nicotiana spp. and Arabidopsis thaliana. Protein extracts were first prepared from leaf tissue of uninfected tobacco plants, and the proteins were visualized with two-dimensional electrophoresis (2-DE). Matching gels were then run using protein extracts of a tobacco plant infected with tobacco mosaic virus (TMV). After visual comparison, the proteins spots that were differentially expressed in infected plant tissues were cut from the gels and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Tandem mass spectrometry data of individual peptides was searched with SEQUEST. Using this approach we demonstrated a successful proof-of-concept experiment by identifying TMV proteins present in the total protein extract. The same procedure was then applied to tobacco plants infected with a laboratory viral isolate of unknown identity. Several of the differentially expressed protein spots were identified as proteins of potato virus X (PVX), thus successfully identifying the causative agent of the uncharacterized viral infection. We believe this demonstrates that HPLC-MS/MS can be used to successfully characterize unknown viruses in infected plants.     

14-3-3 Proteins Interact with FRMD6 and Regulate Its Subcellular Localization in Breast Cancer Cells

The Hippo pathway is evolutionarily conserved from Drosophila to mammals. FRMD6 is one crucial upstream component of the Hippo pathway while its function and regulatory mechanism are largely elusive. We decided to purify the protein complex of FRMD6 to further explore its regulatory mechanism. We established the MCF7 breast cancer cells that stably expressed FRMD6 by retroviruses infection. And we purified the FRMD6-interacting protein complex for further analysis by high performance liquid chromatography-mass spectrometry/mass spectrometry(HPLC- MS/MS). Interestingly, we observed that the major binding partner of FRMD6 is 14-3-3 family of proteins. The interaction between FRMD6 and 14-3-3 proteins was detected by co-immunoprecipitation(CO-IP). The disruption of their interaction resulted in the nuclear localization of FRMD6. Importantly, the T28A mutant of FRMD6 showed stronger tumor suppressor function than wild type(WT) FRMD6. Our results indicate that 14-3-3 proteins tightly regulate the subcellular localization of FRMD6 so as to endow FRMD6 with the tumor suppressor function on breast cancer.     

Chemistry-based functional proteomics to identify novel deubiquitylating enzymes involved in viral infection

Ubiquitylation is a reversible post-translational modification pathway that regulates a variety of cellular processes including protein degradation and trafficking, intracellular localization, DNA repair, immune response and cellcycle progression. Deubiquitylating enzymes (DUBs) can remove the ubiquitin from the modified proteins and reverse the ubiquitylation-induced biological processes; hence it isn't hard to understand that viral pathogens take advantage of the host cell ubiquitin system through disturbing DUBs, for infection and replication. Although accumulated virus-related DUBs have been defined, but how viruses regulate their expression and activities is poor understand because of limitation of technologies. Recently, chemistry-based functional proteomics, which can not only monitor the alteration of abundance but also changes in activity of enzymes, was used to study the function of DUBs involved in virus infection and held much promise. Theses works suggest that chemistry-based functional proteomics is a potent strategy for high throughput screening of virus-related DUBs and exploring their roles in virus infection.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non‐engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene‐specific RNA aptamer, combined with a cell‐permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live‐cell imaging of the endogenous mRNA of β‐actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin‐2, cortactin, and cytoplasmic FMR1‐interacting protein 2. The RNA‐imaging technology and its further optimization might permit live‐cell imaging of any RNA molecules.