Eugene A. Kapp

An aspartyl protease directs malaria effector proteins to the host cell

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. To survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease. Here we show that the protein responsible for cleavage of this motif is plasmepsin V (PMV), an aspartic acid protease located in the endoplasmic reticulum. PMV cleavage reveals the export signal (xE/Q/D) at the amino terminus of cargo proteins. Expression of an identical mature protein with xQ at the N terminus generated by signal peptidase was not exported, demonstrating that PMV activity is essential and linked with other key export events. Identification of the protease responsible for export into erythrocytes provides a novel target for therapeutic intervention against this devastating disease.

Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40–100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.

Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

Overview of Tandem Mass Spectrometry (MS/MS) Database Search Algorithms

Mass spectrometry–based methods for the identification of proteins are fundamental platform technologies for proteomics. One comprehensive approach is to subject trypsinized peptides to tandem mass spectrometry (MS/MS) to obtain detailed structural information. Different strategies are available for interpreting MS/MS data and hence deducing the amino acid sequence of the peptides. The most common method is to use a search algorithm to identify peptides by correlating experimental and theoretical MS/MS data (the latter being generated from possible peptides in the protein sequence database). Identified peptides are collated and protein entries from the sequence database inferred. This unit focuses on the most widely used tandem MS peptide identification search algorithms (commercial and open source), their availability, ease of use, strengths, speed and scoring, as well as their relative sensitivity and specificity. Curr. Protoc. Protein Sci. 49:25.2.1‐25.2.19.

Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-β-mediated Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-β-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using label-free spectral counting. These data reveal that Madin-Darby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-β-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins α6β1, α3β1, α2β1, α5β1, αVβ1, and αVβ3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras/TGF-β-mediated EMT.

Secretome-based proteomic profiling of Ras-transformed MDCK cells reveals extracellular modulators of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenetic process by which epithelial cells lose their basic morphological characteristics such as cell-cell contact and gain mesenchymal properties such as increased motility and invasiveness. To gain insights into proteins released from cells that modulate the EMT process, we compared secretome protein expression profiles of MDCK cells and Ras-transformed MDCK cells (21D1) that stably express oncogenic Ras using 2D-DIGE/LC-MS/MS. Differentially expressed secretome proteins were compared with their corresponding gene expression profiles using the Affymetrix GeneChip system. Down-regulated proteins were predominantly involved with cell-cell contact and cell-matrix adhesion (e.g., desmocollin 2, clusterin, collagen XVII and transforming growth factor-beta induced protein ig-h3), while up-regulated proteins were proteases and factors that promote migration (MMP-1, kallikrein 6, TIMP-1, and S100A4/metastasin). Many of the secretome proteins identified in this study have not been previously identified in the context of EMT and may shed light on the underlying mechanisms associated with this cellular process.

Difference gel electrophoresis analysis of Ras-transformed fibroblast cell-derived exosomes

Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras‐transformed NIH3T3 cells. The vesicular nature and size (30–100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2‐D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras‐transformed NIH3T3 cells. LC‐MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen α‐1 (VI), 14‐3‐3 isoforms, guanine nucleotide‐binding proteins (G proteins), the eukaryotic translation initiation factors elF‐3 γ and elF‐5A accumulated (>2‐fold) in exosomes upon Ras‐induced oncogenic transformation. Significantly, the 10.4‐fold increase in v‐Ha‐Ras p21 protein in exosomes derived from Ras‐transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.

CHOMPER: a bioinformatic tool for rapid validation of tandem mass spectrometry search results associated with high-throughput proteomic strategies.

Current efforts aimed at developing high‐throughput proteomics focus on increasing the speed of protein identification. Although improvements in sample separation, enrichment, automated handling, mass spectrometric analysis, as well as data reduction and database interrogation strategies have done much to increase the quality, quantity and efficiency of data collection, significant bottlenecks still exist. Various separation techniques have been coupled with tandem mass spectrometric (MS/MS) approaches to allow a quicker analysis of complex mixtures of proteins, especially where a high number of unambiguous protein identifications are the exception, rather than the rule. MS/MS is required to provide structural / amino acid sequence information on a peptide and thus allow protein identity to be inferred from individual peptides. Currently these spectra need to be manually validated because: (a) the potential of false positive matches i.e., protein not in database, and (b) observed fragmentation trends may not be incorporated into current MS/MS search algorithms. This validation represents a significant bottleneck associated with high‐throughput proteomic strategies. We have developed CHOMPER, a software program which reduces the time required to both visualize and confirm MS/MS search results and generate post‐analysis reports and protein summary tables. CHOMPER extracts the identification information from SEQUEST MS/MS search result files, reproduces both the peptide and protein identification summaries, provides a more interactive visualization of the MS/MS spectra and facilitates the direct submission of manually validated identifications to a database.

Extracellular remodelling during oncogenic Ras-induced epithelial-mesenchymal transition facilitates MDCK cell migration.

Epithelial-mesenchymal transition (EMT) describes a process whereby immotile epithelial cells escape structural constraints imposed by cellular architecture and acquire a phenotype characteristic of migratory mesenchymal cells. Implicated in carcinoma progression and metastasis, EMT has been the focus of several recent proteomics-based studies aimed at identifying new molecular players. To gain insights into extracellular mediators associated with EMT, we conducted an extensive proteomic analysis of the secretome from MDCK cells following oncogenic Ras-induced EMT (21D1 cells). Using Orbitrap technology and a label-free quantitative approach, differential expression of several secreted modulators were revealed. Proteomic findings were further substantiated by mRNA transcript expression analysis with 71% concordance. MDCK cells undergoing Ras-induced EMT remodel the extracellular matrix (ECM) via diminished expression of basement membrane constituents (collagen type IV and laminin 5), up-regulation of extracellular proteases (MMP-1, kallikreins -6 and -7), and increased production and secretion of ECM constituents (SPARC, collagen type I, fibulins -1 and -3, biglycan, and decorin). Collectively, these findings suggest that hierarchical regulation of a subset of extracellular effectors may coordinate a biological response during EMT that enhances cell motility. Transient silencing of MMP-1 in 21D1 cells via siRNA-mediated knockdown attenuated cell migration. Many of the secretome proteins identified broaden our understanding of the EMT process.

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.