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Molecular Biology of the Cell Jan 2021The adenomatous polyposis coli (APC) tumor suppressor protein is associated with the regulation of Wnt signaling; however, APC also controls other cellular processes...
The adenomatous polyposis coli (APC) tumor suppressor protein is associated with the regulation of Wnt signaling; however, APC also controls other cellular processes including the regulation of cell adhesion and migration. The expression of full-length APC in SW480 colorectal cancer cells (SW480+APC) not only reduces Wnt signaling, but increases membrane E-cadherin and restores cell-cell adhesion. This report describes the effects of full-length, wild-type APC (fl-APC) on cell-cell adhesion genes and p120-catenin isoform switching in SW480 colon cancer cells: fl-APC increased the expression of genes implicated in cell-cell adhesion, whereas the expression of negative regulators of E-cadherin was decreased. Analysis of cell-cell adhesion-related proteins in SW480+APC cells revealed an increase in p120-catenin isoform 3A; similarly, depletion of APC altered the p120-catenin protein isoform profile. Expression of ESRP1 (epithelial splice regulatory protein 1) is increased in SW480+APC cells, and its depletion results in reversion to the p120-catenin isoform 1A phenotype and reduced cell-cell adhesion. The ESRP1 transcript is reduced in primary colorectal cancer, and its expression correlates with the level of APC. Pyrvinium pamoate, which inhibits Wnt signaling, promotes ESRP1 expression. We conclude that re-expression of APC restores the cell-cell adhesion gene and posttranscriptional regulatory programs leading to p120-catenin isoform switching and associated changes in cell-cell adhesion.
Topics: Adenomatous Polyposis Coli Protein; Catenins; Cell Adhesion; Cell Line, Tumor; Colorectal Neoplasms; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Protein Isoforms; RNA, Messenger; RNA-Binding Proteins; Subcellular Fractions; Wnt Signaling Pathway; Delta Catenin
PubMed: 33237836
DOI: 10.1091/mbc.E20-05-0321 -
Nature Communications Apr 2023Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like...
Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.
Topics: Humans; Cell Line, Tumor; Melanoma; Skin Neoplasms; Epithelial-Mesenchymal Transition; Epigenesis, Genetic; Protein Isoforms; Gene Expression Regulation, Neoplastic; COUP Transcription Factor II
PubMed: 37015919
DOI: 10.1038/s41467-023-36967-2 -
Cellular and Molecular Life Sciences :... Jan 2023Dysregulated cell migration and invasion are hallmarks of many disease states. This dysregulated migratory behavior is influenced by the changes in expression of... (Review)
Review
Dysregulated cell migration and invasion are hallmarks of many disease states. This dysregulated migratory behavior is influenced by the changes in expression of aquaporins (AQPs) that occur during pathogenesis, including conditions such as cancer, endometriosis, and arthritis. The ubiquitous function of AQPs in migration of diseased cells makes them a crucial target for potential therapeutics; this possibility has led to extensive research into the specific mechanisms underlying AQP-mediated diseased cell migration. The functions of AQPs depend on a diverse set of variables including cell type, AQP isoform, disease state, cell microenvironments, and even the subcellular localization of AQPs. To consolidate the considerable work that has been conducted across these numerous variables, here we summarize and review the last decade's research covering the role of AQPs in the migration and invasion of cells in diseased states.
Topics: Female; Humans; Aquaporins; Protein Isoforms; Cell Movement; Endometriosis
PubMed: 36682037
DOI: 10.1007/s00018-022-04665-9 -
G3 (Bethesda, Md.) Nov 2022Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read...
Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.
Topics: Humans; Male; Transcriptome; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Protein Isoforms; Sequence Analysis, RNA; Lymphocyte Subsets
PubMed: 36161486
DOI: 10.1093/g3journal/jkac253 -
Communications Biology Aug 2020While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in...
While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.
Topics: Histones; Protein Isoforms; Protein Processing, Post-Translational; Proteins; Single-Cell Analysis; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 32747637
DOI: 10.1038/s42003-020-01132-8 -
International Journal of Molecular... Mar 2022Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin...
Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.
Topics: Angiotensin-Converting Enzyme 2; COVID-19; Humans; Male; Protein Isoforms; SARS-CoV-2; Spermatozoa
PubMed: 35409054
DOI: 10.3390/ijms23073694 -
Oncogene Apr 2023Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain...
Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.
Topics: Humans; ras Proteins; Proto-Oncogene Proteins p21(ras); Mutation; Signal Transduction; Neoplasms; Protein Isoforms
PubMed: 36864243
DOI: 10.1038/s41388-023-02638-1 -
JCI Insight Jun 2023Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a...
Fragile X syndrome is a neurodevelopmental disorder caused by the absence of the mRNA-binding protein fragile X messenger ribonucleoprotein (FMRP). Because FMRP is a highly pleiotropic protein controlling the expression of hundreds of genes, viral vector-mediated gene replacement therapy is viewed as a potential viable treatment to correct the fundamental underlying molecular pathology inherent in the disorder. Here, we studied the safety profile and therapeutic effects of a clinically relevant dose of a self-complementary adeno-associated viral (AAV) vector containing a major human brain isoform of FMRP after intrathecal injection into wild-type and fragile X-KO mice. Analysis of the cellular transduction in the brain indicated primarily neuronal transduction with relatively sparse glial expression, similar to endogenous FMRP expression in untreated wild-type mice. AAV vector-treated KO mice showed recovery from epileptic seizures, normalization of fear conditioning, reversal of slow-wave deficits as measured via electroencephalographic recordings, and restoration of abnormal circadian motor activity and sleep. Further assessment of vector efficacy by tracking and analyzing individual responses demonstrated correlations between the level and distribution of brain transduction and drug response. These preclinical findings further demonstrate the validity of AAV vector-mediated gene therapy for treating the most common genetic cause of cognitive impairment and autism in children.
Topics: Animals; Humans; Mice; Fear; Fragile X Mental Retardation Protein; Mice, Knockout; Protein Isoforms; Seizures
PubMed: 37288657
DOI: 10.1172/jci.insight.169650 -
Angewandte Chemie (International Ed. in... Dec 2019Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and...
Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers, which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive structure-activity relationship and broke ground in the development of selective probes to delineate the isoform-specific functions of Akt kinases.
Topics: Allosteric Regulation; Allosteric Site; Humans; Protein Isoforms; Proto-Oncogene Proteins c-akt; Structure-Activity Relationship
PubMed: 31584233
DOI: 10.1002/anie.201909857 -
ASN Neuro 2022Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocytes and neural stem cells, and their malignant analogues in... (Review)
Review
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocytes and neural stem cells, and their malignant analogues in glioma. Since the discovery of the protein 50 years ago, multiple alternative splice variants of the GFAP gene have been discovered, leading to different GFAP isoforms. In this review, we will describe GFAP isoform expression from gene to protein to network, taking the canonical isoforms GFAPα and the main alternative variant GFAPδ as the starting point. We will discuss the relevance of studying GFAP and its isoforms in disease, with a specific focus on diffuse gliomas.
Topics: Alternative Splicing; Astrocytes; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filaments; Protein Isoforms
PubMed: 35673702
DOI: 10.1177/17590914221102065