Paul Ajuh

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors — the spliceosome — is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry

Recently, we identified proteins that co‐purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5+ gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP‐dependent step. We also show that this complex is required for the second catalytic step of pre‐mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre‐mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre‐mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre‐mRNA splicing activity when added to extracts that have been immunodepleted using anti‐CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non‐snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.

Quantitative Proteomics Using Stable Isotope Labeling with Amino Acids in Cell Culture Reveals Changes in the Cytoplasmic, Nuclear, and Nucleolar Proteomes in Vero Cells Infected with the Coronavirus Infectious Bronchitis Virus

Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting ≥2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-κB- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-κB-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research.

Characterization of carbon nanotube protein corona by using quantitative proteomics

UNLABELLED The protein corona of a nanomaterial is a complex layer of proteins spontaneously and stably formed when the material is exposed to body fluids or intracellular environments. In this study, we utilised stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to characterise the binding of human cellular proteins to two forms of carbon nanoparticles: namely multi-walled carbon nanotubes (MWCNTs) and carbon black (CB). The relative binding efficiency of over 750 proteins to these materials is measured. The data indicate that MWCNTs and CB bind to vastly different sets of proteins. The molecular basis of selectivity in protein binding is investigated. This study is the first large-scale characterisation of protein corona on CNT, providing the biochemical basis for the assessment of the suitability of CNTs as biomedical tools, and as an emerging pollutant. FROM THE CLINICAL EDITOR This team of investigators performed the first large-scale characterization of protein corona on carbon nanotubes, studying 750 proteins and assessing the suitability of CNTs as biomedical tools and as an emerging pollutant.

PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors

In papillary renal cell carcinomas the TFE3 transcription factor becomes fused to the PSF and NonO pre-mRNA splicing factors and most commonly to a protein of unknown function designated PRCC. In this study we have examined the ability of the resulting PRCC–TFE3 and NonO–TFE3 fusions to activate transcription from the plasminogen activator inhibitor-1 (PAI-1) promoter. The results show that only fusion to PRCC enhanced transcriptional activation, indicating that the ability to enhance the level of transcription from endogenous TFE3 promoters is not a consistent feature of TFE3 fusions. In investigations of the normal function of PRCC we observed that PRCC expressed as a green fluorescent fusion protein colocalizes within the nucleus with Sm pre-mRNA splicing factors. It was also found that endogenous PRCC is coimmunoprecipitated by antibodies that recognize a variety of pre-mRNA splicing factors including SC35, PRL1 and CDC5. Association with the cellular splicing machinery is therefore, a common feature of the proteins that become fused to TFE3 in papillary renal cell carcinomas.

Interaction of U-box E3 ligase SNEV with PSMB4, the β7 subunit of the 20 S proteasome

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the proteins sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.

Direct interaction between hnRNP-M and CDC5L/ PLRG1 proteins affects alternative splice site choice

Heterogeneous nuclear ribonucleoprotein‐M (hnRNP‐M) is an abundant nuclear protein that binds to pre‐mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP‐M and the human spliceosome proteins cell division cycle 5‐like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat‐shock stress response. A central region in hnRNP‐M is required for interaction with CDC5L/PLRG1. hnRNP‐M affects both 5′ and 3′ alternative splice site choices, and an hnRNP‐M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno‐E1A mini‐gene substrate.

Novel Blood Collection Method Allows Plasma Proteome Analysis from Single Zebrafish

Zebrafish is an important model organism in biological research. One of the least explored tissues of zebrafish is blood, because the existing methods for isolating blood from this organism are tedious and irreproducible. The small volume of blood collected by these methods also prohibits many biochemical and cytological analyses. This technical obstacle limits the utilization of zebrafish in many applications, particularly in hematological research and plasma biomarker discovery. To overcome this limitation, we have established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a wound. This method consistently recovers more blood than traditional methods. Gel electrophoresis and flow cytometry showed that composition of blood harvested by this method is indistinguishable from traditional methods. The increase in yield enables us to perform biochemical experiments on zebrafish blood. In particular, we have demonstrated that quantitative proteomics can be performed on plasma collected from single zebrafish. Here, we have compared, by using shotgun proteomic analysis, the plasma proteomes of adult male and female zebrafish. Twenty-seven gender-dependent plasma proteins are identified and their biochemical importance discussed. Taken together, this novel technique enables analyses that were previously difficult to perform on zebrafish blood.

A quantitative proteomic analysis of lung epithelial (A549) cells infected with 2009 pandemic influenza A virus using stable isotope labelling with amino acids in cell culture

Influenza A virus is one of the worlds major uncontrolled pathogens, causing seasonal epidemics as well as global pandemics. This was evidenced by the recent emergence and now prevalence of the 2009 swine origin pandemic H1N1 influenza A virus. In this study, quantitative proteomics using stable isotope labelling with amino acids in cell culture was used to investigate the changes in the host cell proteome in cells infected with pandemic H1N1 influenza A virus. The study was conducted in A549 cells that retain properties similar to alveolar cells. Several global pathways were affected, including cell cycle regulation and lipid metabolism, and these could be correlated with recent microarray analyses of cells infected with influenza A virus. Taken together, both quantitative proteomics and transcriptomic approaches can be used to identify potential cellular proteins whose functions in the virus life cycle could be targeted for chemotherapeutic intervention