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Prion Jul 2012The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated... (Review)
Review
The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive 'toolbox' available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP (Sc) . Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.
Topics: Amino Acid Sequence; Amyloid; Animals; Gene Expression; Mice; Molecular Sequence Data; Peptide Termination Factors; Prions; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 22449853
DOI: 10.4161/pri.19214 -
Journal of Clinical Pathology Jul 2019The neurotrophic tyrosine receptor kinase () gene family encodes three tropomyosin receptor kinases (TRKA, TRKB, TRKC) that contribute to central and peripheral nervous... (Review)
Review
The neurotrophic tyrosine receptor kinase () gene family encodes three tropomyosin receptor kinases (TRKA, TRKB, TRKC) that contribute to central and peripheral nervous system development and function. gene fusions are oncogenic drivers of various adult and paediatric tumours. Several methods have been used to detect gene fusions including immunohistochemistry, fluorescence in situ hybridisation, reverse transcriptase polymerase chain reaction, and DNA- or RNA-based next-generation sequencing. For patients with TRK fusion cancer, TRK inhibition is an important therapeutic target. Following the FDA approval of the selective TRK inhibitor, larotrectinib, as well as the ongoing development of multi-kinase inhibitors with activity in TRK fusion cancer, testing for gene fusions should become part of the standard diagnostic process. In this review we discuss the biology of gene fusions, and we present a testing algorithm to aid detection of these gene fusions in clinical practice and guide treatment decisions.
Topics: Algorithms; Humans; Membrane Glycoproteins; Neoplasms; Nerve Growth Factors; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptor, trkB; Receptor, trkC
PubMed: 31072837
DOI: 10.1136/jclinpath-2018-205679 -
Annals of Oncology : Official Journal... Nov 2019The tropomyosin receptor kinase (TRK) family of receptor tyrosine kinases are encoded by NTRK genes and have a role in the development and normal functioning of the... (Review)
Review
The tropomyosin receptor kinase (TRK) family of receptor tyrosine kinases are encoded by NTRK genes and have a role in the development and normal functioning of the nervous system. Since the discovery of an oncogenic NTRK gene fusion in colorectal cancer in 1986, over 80 different fusion partner genes have been identified in a wide array of adult and paediatric tumours, providing actionable targets for targeted therapy. This review describes the normal function and physiology of TRK receptors and the biology behind NTRK gene fusions and how they act as oncogenic drivers in cancer. Finally, an overview of the incidence and prevalence of NTRK gene fusions in various types of cancers is discussed.
Topics: Animals; Gene Fusion; Humans; Neoplasms; Oncogene Proteins, Fusion; Protein Kinases; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Signal Transduction
PubMed: 31738427
DOI: 10.1093/annonc/mdz383 -
The Journal of Pathology Feb 2017ALK oncogenic activation mechanisms were characterized in four conventional spindle-cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which... (Review)
Review
ALK oncogenic activation mechanisms were characterized in four conventional spindle-cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which had ALK genomic perturbations. Constitutively activated ALK oncoproteins were purified by ALK immunoprecipitation and electrophoresis, and were characterized by mass spectrometry. The four conventional IMT had TPM3/4-ALK fusions (two cases) or DCTN1-ALK fusions (two cases), whereas two atypical spindle-cell IMT had TFG-ALK and TPM3-ALK fusion in one case each, and three epithelioid inflammatory myofibroblastic sarcomas had RANBP2-ALK fusions in two cases, and a novel RRBP1-ALK fusion in one case. The epithelioid inflammatory myofibroblastic sarcoma with RRBP1-ALK fusion had cytoplasmic ALK expression with perinuclear accentuation, different from the nuclear membranous ALK localization in epithelioid inflammatory myofibroblastic sarcomas with RANBP2-ALK fusions. Evaluation of three additional uncharacterized epithelioid inflammatory myofibroblastic sarcomas with ALK cytoplasmic/perinuclear- accentuation expression demonstrated RRBP1-ALK fusion in two cases. These studies show that atypical spindle-cell IMT can utilize the same ALK fusion mechanisms described previously in conventional IMT, whereas in clinically aggressive epithelioid inflammatory myofibroblastic sarcoma we identify a novel recurrent ALK oncogenic mechanism, resulting from fusion with the RRBP1 gene. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Topics: Anaplastic Lymphoma Kinase; Carrier Proteins; Humans; Inflammation; Myofibroblasts; Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Sarcoma
PubMed: 27874193
DOI: 10.1002/path.4836 -
Philosophical Transactions of the Royal... Aug 1999The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins... (Review)
Review
The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins move on their way to or from the cell surface. Sorting of proteins within this system is achieved by their selective incorporation into budding vesicles and the specific fusion of these with an appropriate target membrane. Cytosolic coat proteins help to select vesicle contents, while fusion is mediated by membrane proteins termed SNAREs present in both vesicles and target membranes. SNAREs are not the sole determinants of target specificity, but they lie at the heart of the fusion process. The complete set of SNAREs is known in yeast, and analysis of their locations, interactions and functions in vivo gives a comprehensive picture of the traffic routes and the ways in which organelles such as the Golgi apparatus are formed. The principles of protein and lipid sorting revealed by this analysis are likely to apply to a wide variety of eukaryotic cells.
Topics: Adenosine Triphosphatases; Biological Transport; Biomarkers; Endocytosis; Fungal Proteins; Golgi Apparatus; Intracellular Membranes; Membrane Fusion; Membrane Proteins; Membrane Transport Proteins; Nerve Tissue Proteins; Qa-SNARE Proteins; SNARE Proteins; Saccharomyces cerevisiae Proteins; Synaptosomal-Associated Protein 25; Vesicular Transport Proteins
PubMed: 10515003
DOI: 10.1098/rstb.1999.0491 -
Journal of Thoracic Oncology : Official... Apr 2024Variable partners and breakpoints have been reported in patients with ROS1-rearranged NSCLC. Here, we investigated the association of fusion partners and breakpoints...
INTRODUCTION
Variable partners and breakpoints have been reported in patients with ROS1-rearranged NSCLC. Here, we investigated the association of fusion partners and breakpoints with crizotinib efficacy in NSCLCs with common ROS1 fusions.
METHODS
DNA and RNA next-generation sequencing (NGS) and immunohistochemistry were performed to characterize ROS1 fusions.
RESULTS
Using DNA NGS, we identified ROS1 fusions in 210 cases, comprising 171 common (CD74/EZR/TPM3/SDC4/SLC34A2-ROS1) and 39 uncommon (variants identified in <5%) ROS1 fusion cases. DNA NGS detected variable ROS1 genomic breakpoints in common ROS1 fusions, whereas RNA NGS found ROS1 breakpoints mainly occurring in exons 32, 34 and 35, resulting in long (exon 32) and short (exon 34 or 35) ROS1 fusions. ROS1 immunohistochemistry revealed that membranous and cytoplasmic staining was predominant in long ROS1 fusions, whereas cytoplasmic staining was predominant in short ROS1 fusions (p = 0.006). For patients who received first-line crizotinib, median progression-free survival (mPFS) was lower in patients with long ROS1 fusions than those with short ROS1 fusions (8.0 versus 24.0 mo, p = 0.006). Moreover, mPFS for patients with and without TP53 mutations was 8.0 and 19.0 months, respectively (p = 0.159); mPFS for patients with and without BIM deletion polymorphism was 5.0 and 22.0 months, respectively (p = 0.003). When analyzing together with fusion partners, patients with long CD74/SLC34A2-ROS1 fusions were found to have shorter PFS than those with other ROS1, regardless of the presence or absence of TP53 mutations (p < 0.001 and p = 0.002, respectively).
CONCLUSIONS
Long CD74/SLC34A2-ROS1 fusions, which retain transmembrane regions in ROS1 and fusion partners, are associated with poor response to crizotinib independent of TP53 mutations.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Crizotinib; DNA; Lung Neoplasms; Mutation; Oncogene Proteins, Fusion; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; RNA; Sodium-Phosphate Cotransporter Proteins, Type IIb; Tumor Suppressor Protein p53; Histocompatibility Antigens Class II; Antigens, Differentiation, B-Lymphocyte
PubMed: 38070598
DOI: 10.1016/j.jtho.2023.12.009 -
The Journal of Molecular Diagnostics :... Mar 2023The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian...
The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.
Topics: Humans; Receptor, trkA; Protein-Tyrosine Kinases; Canada; Proto-Oncogene Proteins; Neoplasms; High-Throughput Nucleotide Sequencing; Gene Fusion; Sequence Analysis, RNA; Oncogene Proteins, Fusion; Autoantigens; Calmodulin-Binding Proteins; Protein Serine-Threonine Kinases
PubMed: 36586421
DOI: 10.1016/j.jmoldx.2022.12.004 -
Journal of Experimental Botany Mar 2017Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein...
Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.
Topics: Arabidopsis; Arabidopsis Proteins; Fluorescence; Green Fluorescent Proteins; Histones; Recombinant Fusion Proteins; Salicylic Acid
PubMed: 28369601
DOI: 10.1093/jxb/erx031 -
Cancer Genetics Jun 2022Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and...
Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.
Topics: Adaptor Proteins, Signal Transducing; Adult; Biomarkers, Tumor; Carcinogenesis; Child; Cytoskeletal Proteins; Gene Fusion; Humans; Neoplasms; Oncogene Proteins, Fusion; Receptor, trkA
PubMed: 35366592
DOI: 10.1016/j.cancergen.2022.03.001 -
Molecular Biology of the Cell Aug 2012Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable...
Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.
Topics: Amino Acid Transport Systems; Cell Nucleus; Gene Knockout Techniques; Green Fluorescent Proteins; HSP40 Heat-Shock Proteins; Heat-Shock Proteins; Heat-Shock Response; Karyopherins; Microscopy, Fluorescence; Molecular Chaperones; Nuclear Localization Signals; Phenotype; Prions; Protein Binding; Protein Multimerization; Protein Transport; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 22718905
DOI: 10.1091/mbc.E12-03-0194