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Cells Apr 2022Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long...
Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long isoform called is concomitantly expressed with . Once translated, L-VEGF is proteolytically cleaved to generate N-VEGF and VEGF-A. Interestingly, while VEGF-A is secreted and affects the surrounding cells, N-VEGF is mobilized to the nucleus. This suggests that N-VEGF participates in transcriptional response to hypoxia. In this study, we performed a series of complementary experiments to examine the functional role of N-VEGF. Strikingly, we found that the mere expression of N-VEGF followed by its hypoxia-independent mobilization to the nucleus was sufficient to induce key genes associated with angiogenesis, such as isoforms, as well as genes associated with cell survival under hypoxia. Complementarily, when was genetically depleted, key hypoxia-induced genes were downregulated and cells were significantly susceptible to hypoxia-mediated apoptosis. This is the first report of N-VEGF serving as an autoregulatory arm of VEGF-A. Further experiments will be needed to determine the role of N-VEGF in cancer and embryogenesis.
Topics: Apoptosis; Cell Hypoxia; Humans; Hypoxia; Neovascularization, Pathologic; Protein Isoforms; Vascular Endothelial Growth Factor A
PubMed: 35455969
DOI: 10.3390/cells11081289 -
RATTLE: reference-free reconstruction and quantification of transcriptomes from Nanopore sequencing.Genome Biology Jul 2022Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping...
Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping reads to a reference genome, which precludes the study of species with a partial or missing reference or the identification of disease-specific transcripts not readily identifiable from a reference. We present RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore reads. Using simulated data and experimental data from isoform spike-ins, human tissues, and cell lines, we show that RATTLE accurately determines transcript sequences and their abundances, and shows good scalability with the number of transcripts.
Topics: High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing; Nanopores; Protein Isoforms; Transcriptome
PubMed: 35804393
DOI: 10.1186/s13059-022-02715-w -
Cell Death & Disease Mar 2024The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote... (Review)
Review
The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.
Topics: Humans; Ubiquitination; Protein Isoforms; Gastrointestinal Neoplasms; Carcinogenesis; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases
PubMed: 38453895
DOI: 10.1038/s41419-024-06575-z -
PloS One 2022Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by...
Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. Here, by characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, we show that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. We propose that expression dynamics and competitive interactions determine isoform participation in these processes. We propose that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation.
Topics: Animals; Brain; CRISPR-Cas Systems; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Eye; Gene Editing; Genotype; LIM Domain Proteins; Membrane Proteins; Phenotype; Protein Isoforms; RNA Interference; RNA, Small Interfering; Signal Transduction
PubMed: 35148314
DOI: 10.1371/journal.pone.0262328 -
Nature Communications Sep 2023Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the...
Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.
Topics: Humans; Proteomics; Protein Isoforms; Biomedical Research; Carcinoma, Hepatocellular; Liver Neoplasms
PubMed: 37726316
DOI: 10.1038/s41467-023-41558-2 -
Cells Apr 2021p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53...
p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all- retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French-American-British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.
Topics: Adult; Aged; Aged, 80 and over; Blast Crisis; Cell Differentiation; Cell Line, Tumor; Epigenesis, Genetic; Female; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Protein Isoforms; Tumor Suppressor Protein p53; Valproic Acid
PubMed: 33917201
DOI: 10.3390/cells10040833 -
Nature Protocols Feb 2021Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not...
Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.
Topics: Animals; Blastocyst; Blastomeres; DNA; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Embryonic Development; Female; Immunoblotting; Mice; Nucleic Acids; Protein Isoforms; RNA, Messenger
PubMed: 33452502
DOI: 10.1038/s41596-020-00449-2 -
Scientific Reports Jan 2020The advent of RNA-seq technologies has switched the paradigm of genetic analysis from a genome to a transcriptome-based perspective. Alternative splicing generates...
The advent of RNA-seq technologies has switched the paradigm of genetic analysis from a genome to a transcriptome-based perspective. Alternative splicing generates functional diversity in genes, but the precise functions of many individual isoforms are yet to be elucidated. Gene Ontology was developed to annotate gene products according to their biological processes, molecular functions and cellular components. Despite a single gene may have several gene products, most annotations are not isoform-specific and do not distinguish the functions of the different proteins originated from a single gene. Several approaches have tried to automatically annotate ontologies at the isoform level, but this has shown to be a daunting task. We have developed ISOGO (ISOform + GO function imputation), a novel algorithm to predict the function of coding isoforms based on their protein domains and their correlation of expression along 11,373 cancer patients. Combining these two sources of information outperforms previous approaches: it provides an area under precision-recall curve (AUPRC) five times larger than previous attempts and the median AUROC of assigned functions to genes is 0.82. We tested ISOGO predictions on some genes with isoform-specific functions (BRCA1, MADD,VAMP7 and ITSN1) and they were coherent with the literature. Besides, we examined whether the main isoform of each gene -as predicted by APPRIS- was the most likely to have the annotated gene functions and it occurs in 99.4% of the genes. We also evaluated the predictions for isoform-specific functions provided by the CAFA3 challenge and results were also convincing. To make these results available to the scientific community, we have deployed a web application to consult ISOGO predictions (https://biotecnun.unav.es/app/isogo). Initial data, website link, isoform-specific GO function predictions and R code is available at https://gitlab.com/icassol/isogo.
Topics: Algorithms; Alternative Splicing; Gene Ontology; Humans; Molecular Sequence Annotation; Open Reading Frames; Protein Isoforms
PubMed: 31974522
DOI: 10.1038/s41598-020-57974-z -
The Journal of Biological Chemistry Nov 2020The role of proliferation-associated protein 2G4 (PA2G4), alternatively known as ErbB3-binding protein 1 (EBP1), in cancer has become apparent over the past 20 years.... (Review)
Review
The role of proliferation-associated protein 2G4 (PA2G4), alternatively known as ErbB3-binding protein 1 (EBP1), in cancer has become apparent over the past 20 years. PA2G4 expression levels are correlated with prognosis in a range of human cancers, including neuroblastoma, cervical, brain, breast, prostate, pancreatic, hepatocellular, and other tumors. There are two PA2G4 isoforms, PA2G4-p42 and PA2G4-p48, and although both isoforms of PA2G4 regulate cellular growth and differentiation, these isoforms often have opposing roles depending on the context. Therefore, PA2G4 can function either as a contextual tumor suppressor or as an oncogene, depending on the tissue being studied. However, it is unclear how distinct structural features of the two PA2G4 isoforms translate into different functional outcomes. In this review, we examine published structures to identify important structural and functional components of PA2G4 and consider how they may explain its crucial role in the malignant phenotype. We will highlight the lysine-rich regions, protein-protein interaction sites, and post-translational modifications of the two PA2G4 isoforms and relate these to the functional cellular role of PA2G4. These data will enable a better understanding of the function and structure relationship of the two PA2G4 isoforms and highlight the care that will need to be undertaken for those who wish to conduct isoform-specific structure-based drug design campaigns.
Topics: Adaptor Proteins, Signal Transducing; Humans; Neoplasm Proteins; Neoplasms; Protein Isoforms; RNA-Binding Proteins; Structure-Activity Relationship
PubMed: 32952126
DOI: 10.1074/jbc.REV120.014293 -
ELife Dec 2022Recently developed methods to predict three-dimensional protein structure with high accuracy have opened new avenues for genome and proteome research. We explore a new...
Recently developed methods to predict three-dimensional protein structure with high accuracy have opened new avenues for genome and proteome research. We explore a new hypothesis in genome annotation, namely whether computationally predicted structures can help to identify which of multiple possible gene isoforms represents a functional protein product. Guided by protein structure predictions, we evaluated over 230,000 isoforms of human protein-coding genes assembled from over 10,000 RNA sequencing experiments across many human tissues. From this set of assembled transcripts, we identified hundreds of isoforms with more confidently predicted structure and potentially superior function in comparison to canonical isoforms in the latest human gene database. We illustrate our new method with examples where structure provides a guide to function in combination with expression and evolutionary evidence. Additionally, we provide the complete set of structures as a resource to better understand the function of human genes and their isoforms. These results demonstrate the promise of protein structure prediction as a genome annotation tool, allowing us to refine even the most highly curated catalog of human proteins. More generally we demonstrate a practical, structure-guided approach that can be used to enhance the annotation of any genome.
Topics: Humans; Transcriptome; Molecular Sequence Annotation; Protein Isoforms; Genome; Sequence Analysis, RNA
PubMed: 36519529
DOI: 10.7554/eLife.82556