Alex Ignatchenko

Comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology

Placental abnormalities are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting ∼5% of all pregnancies. An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities, but there have been no systematic attempts to assess their molecular similarities or differences. We collected protein and mRNA expression data through shot‐gun proteomics and microarray expression analysis of the highly vascular exchange region, microdissected from the human and mouse near‐term placenta. Over 7000 ortholog genes were detected with 70% co‐expressed in both species. Close to 90% agreement was found between our human proteomic results and 1649 genes assayed by immunohistochemistry for expression in the human placenta in the Human Protein Atlas. Interestingly, over 80% of genes known to cause placental phenotypes in mouse are co‐expressed in human. Several of these phenotype‐associated proteins form a tight protein–protein interaction network involving 15 known and 34 novel candidate proteins also likely important in placental structure and/or function. The entire data are available as a web‐accessible database to guide the informed development of mouse models to study human disease.

In-Depth Proteomic Analyses of Direct Expressed Prostatic Secretions

It is expected that clinically obtainable fluids that are proximal to organs contain a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue and thus represent potential sources for biomarker discovery, investigation of tissue-specific biology, and assay development. The prostate gland secretes many proteins into a prostatic fluid that combines with seminal vesicle fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed prostatic secretion (EPS) fluids. In the current study, MudPIT-based proteomics was applied to EPS obtained from nine men with prostate cancer and resulted in the confident identification of 916 unique proteins. Systematic bioinformatics analyses using publicly available microarray data of 21 human tissues (Human Gene Atlas), the Human Protein Atlas database, and other published proteomics data of shed/secreted proteins were performed to systematically analyze this comprehensive proteome. Therefore, we believe this data will be a valuable resource for the research community to study prostate biology and potentially assist in the identification of novel prostate cancer biomarkers. To further streamline this process, the entire data set was deposited to the Tranche repository for use by other researchers.

In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome.

Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery.

Translational Analysis of Mouse and Human Placental Protein and mRNA Reveals Distinct Molecular Pathologies in Human Preeclampsia

Preeclampsia (PE) adversely impacts ∼5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms

The freshwater planarian Schmidtea mediterranea has been used in research for over 100 years, and is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. Exteriorly, planarians are covered in mucous secretions of unknown composition, implicated in locomotion, predation, innate immunity, and substrate adhesion. Although the planarian genome has been sequenced, it remains mostly unannotated, challenging both genomic and proteomic analyses. The goal of the current study was to annotate the proteome of the whole planarian and its mucous fraction. The S. mediterranea proteome was analyzed via mass spectrometry by using multidimensional protein identification technology with whole-worm tryptic digests. By using a proteogenomics approach, MS data were searched against an in silico translated planarian transcript database, and by using the Swiss-Prot BLAST algorithm to identify proteins similar to planarian queries. A total of 1604 proteins were identified. The mucous subproteome was defined through analysis of a mucous trail fraction and an extract obtained by treating whole worms with the mucolytic agent N-acetylcysteine. Gene Ontology analysis confirmed that the mucous fractions were enriched with secreted proteins. The S. mediterranea proteome is highly similar to that predicted for the trematode Schistosoma mansoni associated with intestinal schistosomiasis, with the mucous subproteome particularly highly conserved. Remarkably, orthologs of 119 planarian mucous proteins are present in human mucosal secretions and tear fluid. We suggest planarians have potential to be a model system for the characterization of mucous protein function and relevant to parasitic flatworm infections and diseases underlined by mucous aberrancies, such as cystic fibrosis, asthma, and other lung diseases.

Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP).

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca2+ release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1–4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca2+ release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.

Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media

Secreted proteins are of tremendous biological interest since they can act as ligands for receptors to activate downstream signalling cascades or be used as biomarkers if altered abundance is correlated with a specific pathological state. Proteins can be secreted either as soluble molecules or as part of extracellular vesicles (i.e., exosomes or microvesicles). The complete proteomic profiling of secretomes requires analysis of secreted proteins and extracellular vesicles. Hence, the method described here enriches for microvesicles, exosomes, and secreted proteins from conditioned media using differential centrifugation. The three fractions are then analyzed by mass spectrometry-based proteomics for in-depth characterization and comparison of the protein secretome of cell lines.

Multidimensional protein identification technology analysis highlights mitoxantrone-induced expression modulations in the primary prostate cancer cell proteome

Chemotherapeutic agents as they are used today have limited effectiveness against prostate cancer, but may potentially be used in new combinations with more efficacious results. Mitoxantrone, used for palliation of prostate cancer, has recently been found by our group to improve the susceptibility of primary prostate cancer cells to killing through the Fas‐mediated death pathway. Here we used a shotgun proteomics approach to first profile the entire prostate cancer proteome and then identify specific factors involved in this mitoxantrone response. Peptides derived from primary prostate cancer cells treated with or without 100 nM mitoxantrone were analyzed by multidimensional protein identification technology (MudPIT). Strict limits and data filtering hierarchies were applied to identify proteins with high confidence. We identified 1498 proteins belonging to the prostate cancer proteome, 83 of which were significantly upregulated and 27 of which were markedly downregulated following mitoxantrone treatment. These proteins perform diverse functions, including ceramide production, tumour suppression, and oxidative reduction. Detailed proteomic analyses of prostate cancer cells and their response to mitoxantrone will further our understanding of its mechanisms of action. Identification of proteins influenced by treatment with mitoxantrone or other compounds may lead to the development of more effective drug combinations against prostate cancer.