Global network analysis in Schizosaccharomyces pombe reveals three distinct consequences of the common 1-kb deletion causing juvenile CLN3 disease.

Juvenile CLN3 disease is a recessively inherited paediatric neurodegenerative disorder, with most patients homozygous for a 1-kb intragenic deletion in CLN3. The btn1 gene is the Schizosaccharomyces pombe orthologue of CLN3. Here, we have extended the use of synthetic genetic array (SGA) analyses to delineate functional signatures for two different disease-causing mutations in addition to complete deletion of btn1. We show that genetic-interaction signatures can differ for mutations in the same gene, which helps to dissect their distinct functional effects. The mutation equivalent to the minor transcript arising from the 1-kb deletion (btn1102-208del) shows a distinct interaction pattern. Taken together, our results imply that the minor 1-kb deletion transcript has three consequences for CLN3: to both lose and retain some inherent functions and to acquire abnormal characteristics. This has particular implications for the therapeutic development of juvenile CLN3 disease. In addition, this proof of concept could be applied to conserved genes for other mendelian disorders or any gene of interest, aiding in the dissection of their functional domains, unpacking the global consequences of disease pathogenesis, and clarifying genotype-phenotype correlations. In doing so, this detail will enhance the goals of personalised medicine to improve treatment outcomes and reduce adverse events.

Author Correction: Towards a data-integrated cell.

The original version of this Article contained an error in the spelling of the author Harry Hemingway, which was incorrectly given as Harry Hemmingway. This has been corrected in both the PDF and HTML versions of the Article.

Towards a data-integrated cell.

We are increasingly accumulating molecular data about a cell. The challenge is how to integrate them within a unified conceptual and computational framework enabling new discoveries. Hence, we propose a novel, data-driven concept of an integrated cell, iCell. Also, we introduce a computational prototype of an iCell, which integrates three omics, tissue-specific molecular interaction network types. We construct iCells of four cancers and the corresponding tissue controls and identify the most rewired genes in cancer. Many of them are of unknown function and cannot be identified as different in cancer in any specific molecular network. We biologically validate that they have a role in cancer by knockdown experiments followed by cell viability assays. We find additional support through Kaplan-Meier survival curves of thousands of patients. Finally, we extend this analysis to uncover pan-cancer genes. Our methodology is universal and enables integrative comparisons of diverse omics data over cells and tissues.

GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling.

High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).

Systematic Identification of Oncogenic EGFR Interaction Partners.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) that-once activated upon ligand binding-leads to receptor dimerization, recruitment of protein complexes, and activation of multiple signaling cascades. The EGFR is frequently overexpressed or mutated in various cancers leading to aberrant signaling and tumor growth. Hence, identification of interaction partners that bind to mutated EGFR can help identify novel targets for drug discovery. Here, we used a systematic approach to identify novel proteins that are involved in cancerous EGFR signaling. Using a combination of high-content imaging and a mammalian membrane two-hybrid protein-protein interaction method, we identified eight novel interaction partners of EGFR, of which half strongly interacted with oncogenic, hyperactive EGFR variants. One of these, transforming acidic coiled-coil proteins (TACC) 3, stabilizes EGFR on the cell surface, which results in an increase in downstream signaling via the mitogen-activated protein kinase and AKT pathway. Depletion of TACC3 from cells using small hairpin RNA (shRNA) knockdown or small-molecule targeting reduced mitogenic signaling in non-small cell lung cancer cell lines, suggesting that targeting TACC3 has potential as a new therapeutic approach for non-small cell lung cancer.

Application guide for omics approaches to cell signaling.

Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations.

In silico prediction of physical protein interactions and characterization of interactome orphans.

Protein-protein interactions (PPIs) are useful for understanding signaling cascades, predicting protein function, associating proteins with disease and fathoming drug mechanism of action. Currently, only ∼ 10% of human PPIs may be known, and about one-third of human proteins have no known interactions. We introduce FpClass, a data mining-based method for proteome-wide PPI prediction. At an estimated false discovery rate of 60%, we predicted 250,498 PPIs among 10,531 human proteins; 10,647 PPIs involved 1,089 proteins without known interactions. We experimentally tested 233 high- and medium-confidence predictions and validated 137 interactions, including seven novel putative interactors of the tumor suppressor p53. Compared to previous PPI prediction methods, FpClass achieved better agreement with experimentally detected PPIs. We provide an online database of annotated PPI predictions (http://ophid.utoronto.ca/fpclass/) and the prediction software (http://www.cs.utoronto.ca/~juris/data/fpclass/).

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation.

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase-specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APC(Cdh1)) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation.

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

CHIP-MYTH: a novel interactive proteomics method for the assessment of agonist-dependent interactions of the human ß2-adrenergic receptor.

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαßγ, into Gα and Gßγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.

Investigation of membrane protein interactions using the split-ubiquitin membrane yeast two-hybrid system.

Proteins are generally organized into molecular complexes, in which multiple interaction partners collaborate to carry out cellular processes. Thus, techniques to map protein-protein interactions have become pivotal for biological studies of as yet uncharacterized proteins. Investigation of interaction partners of membrane proteins is of special interest, as they play a major role in cellular processes and are often directly linked to human diseases. Owing to their hydrophobic nature, however, it has proven difficult to study their interaction partners. To circumvent this problem, a yeast-based genetic technology for the in vivo detection of membrane protein interactions, the split-ubiquitin membrane yeast two-hybrid (MYTH) system, has been developed. MYTH allows for detection of both stable and transient interactions and can be applied to large- and small-scale screens. It uses the split-ubiquitin approach, in which the reconstitution of two ubiquitin halves is mediated by a specific protein-protein interaction. Briefly, the bait membrane protein is fused to the C-terminal half of ubiquitin and an artificial transcription factor. The mutated N-terminal moiety of ubiquitin is fused to the prey protein. Upon interaction of bait and prey proteins, ubiquitin is reconstituted and further recognized by ubiquitin-specific proteases, which subsequently cleave off the transcription factor, thus resulting in reporter gene activation. To date, MYTH has been successfully applied to study interactions of membrane proteins from various organisms and has only recently been adapted for the identification of interaction partners of mammalian receptor tyrosine kinases.

Using yeast as a model to study membrane proteins.

PURPOSE OF REVIEW: Many cellular processes are controlled via either stable or transient protein-protein interactions (PPIs). Protein complexes are 'molecular machines' in which multiple interactive partners carry out various cellular functions. Given that almost a third of the proteome consists of membrane proteins and that more than 50% of currently available drugs are targeted toward them, investigation of membrane protein complexes has taken center stage over the past years. Thus, gaining an in-depth understanding of PPI networks will give us more insight into the functional relationship as well as downstream effectors of protein complexes, hence opening strategies for new drug target definitions. RECENT FINDINGS: Studying membrane proteins in yeast has recently been applied to many different classes of proteins with diverse functions and structures including membrane transporters. Techniques such as the split-ubiquitin membrane yeast two-hybrid or variants of the protein-fragment complementation assay have been successfully applied to both large-scale genome-wide screens and as smaller-scale PPI studies in a reliable and robust fashion. SUMMARY: Yeast-based methods to study membrane PPI in vivo offer a powerful tool for the investigation of protein complexes from various organisms, including mammals. The investigation of global protein maps will serve as a foundation for mechanistic and quantitative studies of poorly characterized gene products and disease-associated proteins. Identification of PPIs is also of great interest for drug discovery as many human diseases result from abnormal PPIs.

Interactive proteomics research technologies: recent applications and advances.

Proteins rarely exert their function alone. They normally function in multiprotein complexes that play central roles in all biological functions. Thus, it is not surprising that the investigation of protein-protein interactions on a global scale, of so-called interactomes, has become crucial to modern molecular biology. Dissecting partners in protein complexes gives insight into their molecular function and can help in understanding disease-related mechanisms, ultimately resulting in better drug target definition. A variety of methods exist to unravel protein interaction circuitries and recently, significant progress has been made in adapting these tools for the generation of large-scale interaction datasets. Here, we present an overview of the latest advances and applications of interactive proteomics research technologies.

Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast.

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.

Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly.

We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.

Cdk1/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression.

Triacylglycerols (TGs) serve essential cellular functions as reservoirs for energy substrates (fatty acids) and membrane lipid precursors (diacylglycerols and fatty acids). Here we show that the major yeast TG lipase Tgl4, the functional ortholog of murine adipose TG lipase ATGL, is phosphorylated and activated by cyclin-dependent kinase 1 (Cdk1/Cdc28). Phospho-Tgl4-catalyzed lipolysis contributes to early bud formation in late G1 phase of the cell cycle. Conversely, lack of lipolysis delays bud formation and cell-cycle progression. In the absence of beta-oxidation, lipolysis-derived metabolites are thus required to support cellular growth. TG homeostasis is the only metabolic process identified as yet that is directly regulated by Cdk1/Cdc28-dependent phosphorylation of key anabolic and catabolic enzymes, highlighting the importance of FA storage and mobilization during the cell cycle. Our data provide evidence for a direct link between cell-cycle-regulatory kinases and TG degradation and suggest a general mechanism for coordinating membrane synthesis with cell-cycle progression.

SLipid-induced cell dysfunction and cell death: lessons from yeast.

The 2001 Nobel Prize in Medicine, awarded to two yeast researchers for contributions to understanding the eukaryotic cell cycle, spotlighted yeast as an experimental model system in biomedical research. Major discoveries of molecular processes underlying lipid and biomembrane biogenesis were first made in yeast: secretory pathways, vesicle and membrane fusion, and the unfolded protein response. The discovery of programmed cell death that is conserved at multiple levels (quite intriguing for a unicellular organism), and energy metabolism controlled by adenosine monophosphate-activated protein kinases, mitogen-activated protein kinase signaling pathways, and the target of rapamycin (TOR) pathway (originally discovered in yeast)-all refer to functional and structural similarities with mammalian cells beyond the mere metabolic level. This article reviews recently uncovered aspects of fatty acid-associated malfunctions and lipotoxicity in yeast that may aid in understanding the molecular basis of lipid-associated disorders in mammals.

Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast.

Storage and degradation of triglycerides are essential processes to ensure energy homeostasis and availability of precursors for membrane lipid synthesis. Recent evidence suggests that an emerging class of enzymes containing a conserved patatin domain are centrally important players in lipid degradation. Here we describe the identification and characterization of a major triglyceride lipase of the adipose triglyceride lipase/Brummer family, Tgl4, in the yeast Saccharomyces cerevisiae. Elimination of Tgl4 in a tgl3 background led to fat yeast, rendering growing cells unable to degrade triglycerides. Tgl4 and Tgl3 lipases localized to lipid droplets, independent of each other. Serine 315 in the GXSXG lipase active site consensus sequence of the patatin domain of Tgl4 is essential for catalytic activity. Mouse adipose triglyceride lipase (which also contains a patatin domain but is otherwise highly divergent in primary structure from any yeast protein) localized to lipid droplets when expressed in yeast, and significantly restored triglyceride breakdown in tgl4 mutants in vivo. Our data identify yeast Tgl4 as a functional ortholog of mammalian adipose triglyceride lipase.

The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae.

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.