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Expert Review of Proteomics Aug 2016Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems.... (Review)
Review
INTRODUCTION
Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood.
AREAS COVERED
Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart. Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets.
Topics: Biomarkers; Heart Diseases; Humans; Mass Spectrometry; Protein Isoforms; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 27448560
DOI: 10.1080/14789450.2016.1209414 -
BMC Bioinformatics May 2021Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental... (Review)
Review
BACKGROUND
Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are short.
RESULTS
Here we use simulated benchmarking data that reflects many properties of real data, including polymorphisms, intron signal and non-uniform coverage, allowing for systematic comparative analyses of isoform quantification accuracy and its impact on differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a simple approach is included as a baseline control.
CONCLUSIONS
Salmon, kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform dramatically better than the simple approach. We determine the structural parameters with the greatest impact on quantification accuracy to be length and sequence compression complexity and not so much the number of isoforms. The effect of incomplete annotation on performance is also investigated. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification and isoform level DE should still be employed selectively.
Topics: Gene Expression Profiling; Protein Isoforms; RNA-Seq; Sequence Analysis, RNA; Transcriptome
PubMed: 34034652
DOI: 10.1186/s12859-021-04198-1 -
Journal of Proteome Research Mar 2023Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms...
Functional differentiation of the two isoforms of the protein-serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is an unsettled area of research. The isoforms are highly similar in structure and are largely redundant, though there is also evidence for specific roles. Identification of isoform-specific protein interactors may elucidate the differences in function and provide insight into isoform-selective regulation. We therefore sought to identify novel GSK-3 interaction partners and to examine differences in the interactomes of the two isoforms using both affinity purification and proximity-dependent biotinylation (BioID) mass spectrometry methods. While the interactomes of the two isomers are highly similar in HEK293 cells, BioID in HeLa cells yielded a variety of preys that are preferentially associated with one of the two isoforms. DCP1B, which favored GSK-3α, and MISP, which favored GSK-3β, were evaluated for reciprocal interactions. The differences in interactions between isoforms may help in understanding the distinct functions and regulation of the two isoforms as well as offer avenues for the development of isoform-specific strategies.
Topics: Humans; Glycogen Synthase Kinase 3; HeLa Cells; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Protein Isoforms
PubMed: 36779422
DOI: 10.1021/acs.jproteome.2c00825 -
Briefings in Bioinformatics Jul 2023Neurodegenerative diseases (NDs) usually connect with aggregation and molecular interactions of pathological proteins. The integration of accumulative data from clinical...
Neurodegenerative diseases (NDs) usually connect with aggregation and molecular interactions of pathological proteins. The integration of accumulative data from clinical and biomedical research will allow for the excavation of pathological proteins and related interactors. It is also important to systematically study their interacting proteins in order to find more related proteins and potential therapeutic targets. Understanding binding regions in protein interactions will help functional proteomics and provide an alternative method for predicting novel interactions. This study integrated data from biomedical research to achieve systematic mining and analysis of pathogenic proteins and their interaction network. A workflow has been built as a solution for the collective information of proteins involved in NDs, related protein-protein interactions (PPIs) and interactive visualizations. It also included protein isoforms and mapped them in a disease-related PPI network to illuminate the impact of alternative splicing on protein binding. The interacting proteins enriched by diseases and biological processes (BPs) revealed possible regulatory modules. A high-resolution network with structural affinity information was generated. Finally, Neurodegenerative Disease Atlas (NDAtlas) was constructed with an interactive and intuitive view of protein docking with 3D molecular graphics beyond the traditional 2D network. NDAtlas is available at http://bis.zju.edu.cn/ndatlas.
Topics: Humans; Protein Binding; Protein Interaction Mapping; Neurodegenerative Diseases; Databases, Protein; Protein Isoforms; Protein Interaction Maps
PubMed: 37350526
DOI: 10.1093/bib/bbad237 -
Biochimica Et Biophysica Acta Jan 2015Sarcomeric protein isoforms are mainly governed by alternative promoter-driven expression, distinct gene expression, gene mutation and alternative mRNA splicing. The... (Review)
Review
Sarcomeric protein isoforms are mainly governed by alternative promoter-driven expression, distinct gene expression, gene mutation and alternative mRNA splicing. The transitions of sarcomeric proteins have been implicated to play a role in the onset and development of human heart failure. In this mini-review, we summarized isoform transitions of several most widely examined sarcomeric proteins including myosin, actin, troponin, tropomyosin, titin and myosin binding protein-C, and the consequence of these abnormal isoform transitions. Even though the isoform transitions of sarcomeric proteins have been described in individual sarcomeric protein reviews, no concise summary of these results has been presented previously. This review is intended to fill this gap and discuss possible future perspectives.
Topics: Animals; Heart Failure; Humans; Muscle Proteins; Myocardium; Protein Isoforms; Sarcomeres
PubMed: 25446994
DOI: 10.1016/j.bbadis.2014.11.003 -
Cellular Signalling Oct 2014CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor... (Review)
Review
CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor microenvironment. CD44 contains two variable regions encoded by variable exons. Alternative splicing, which is often deregulated in cancer, can produce various isoforms of CD44 with properties that may have different tissue specific effects and therefore even diverse effects on cancer progression. This review summarizes and puts together all major regulators of alternative splicing of CD44 in cancer that have been documented so far and that have an experimentally proved effect on CD44 isoform switching. It is important to better understand the mechanisms of alternative splicing of CD44, where all the variability of CD44 originates, to be able to explain the isoform switching and occurrence of variant isoforms of CD44 (CD44v) in cancer.
Topics: Alternative Splicing; Epithelial-Mesenchymal Transition; Humans; Hyaluronan Receptors; Neoplasms; Neoplastic Stem Cells; Protein Isoforms
PubMed: 25025570
DOI: 10.1016/j.cellsig.2014.07.011 -
Blood Advances Dec 2023Somatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset...
Somatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset inflammatory disease associated with bone marrow failure and high mortality. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous mutations affecting methionine-41 (M41), leading to the expression of UBA1M41T, UBA1M41V, or UBA1M41L and globally reduced protein polyubiquitination. Here, we used CRISPR-Cas9 to engineer isogenic 32D mouse myeloid cell lines expressing hemizygous Uba1WT or Uba1M41L from the endogenous locus. Consistent with prior analyses of patients with VEXAS syndrome samples, hemizygous Uba1M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform, reduced polyubiquitination, abnormal cytoplasmic vacuoles, and increased production of interleukin-1β and inflammatory chemokines. Vacuoles in Uba1M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Uba1M41L cells were more sensitive to the UBA1 inhibitor TAK243. TAK243 treatment promoted apoptosis in Uba1M41L cells and led to preferential loss of Uba1M41L cells in competition assays with Uba1WT cells. Knock-in of a TAK243-binding mutation, Uba1A580S, conferred TAK243 resistance. In addition, overexpression of catalytically active UBA1b in Uba1M41L cells restored polyubiquitination and increased TAK243 resistance. Altogether, these data indicate that loss of UBA1b underlies a key biochemical phenotype associated with VEXAS syndrome and renders cells with reduced UBA1 activity vulnerable to targeted UBA1 inhibition. Our Uba1M41L knock-in cell line is a useful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.
Topics: Animals; Mice; Humans; Myeloid Cells; Myeloid Progenitor Cells; Lysosomes; Protein Isoforms
PubMed: 38091008
DOI: 10.1182/bloodadvances.2023010531 -
Nature Chemical Biology Jun 2020CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly...
CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.
Topics: Animals; Binding Sites; Circadian Clocks; Cryptochromes; Fibroblasts; HEK293 Cells; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Male; Mice, Knockout; Models, Molecular; Protein Binding; Protein Conformation; Protein Isoforms; Thermodynamics
PubMed: 32231341
DOI: 10.1038/s41589-020-0505-1 -
PloS One 2022TMPRSS6 is a type II transmembrane serine protease involved in iron homeostasis expressed as 4 isoforms in humans. TMPRSS6 isoform 2 downregulates hepcidin production by...
TMPRSS6 is a type II transmembrane serine protease involved in iron homeostasis expressed as 4 isoforms in humans. TMPRSS6 isoform 2 downregulates hepcidin production by cleaving hemojuvelin and other surface proteins of hepatocytes. The functions of catalytically impaired isoforms 3 and 4 are still unknown. Here we demonstrate that TMPRSS6 isoforms 3 and 4 reduce the proteolytic activity of isoform 2 and uncover the ability of isoforms to interact. Moreover, we identified 49 potential protein partners common to TMPRSS6 isoforms, including TfR1, known to be involved in iron regulation. By co-expressing TMPRSS6 and TfR1, we show that TfR1 is cleaved and shed from the cell surface. Further, we demonstrate that TMPRSS6 isoforms 3 and 4 behave as dominant negative.
Topics: Cell Membrane; Hepcidins; Humans; Iron; Membrane Proteins; Protein Isoforms; Serine Endopeptidases
PubMed: 36044454
DOI: 10.1371/journal.pone.0273825 -
International Journal of Molecular... Nov 2016Although it is one of the most studied proteins, p53 continues to be an enigma. This protein has numerous biological functions, possesses intrinsically disordered... (Review)
Review
Although it is one of the most studied proteins, p53 continues to be an enigma. This protein has numerous biological functions, possesses intrinsically disordered regions crucial for its functionality, can form both homo-tetramers and isoform-based hetero-tetramers, and is able to interact with many binding partners. It contains numerous posttranslational modifications, has several isoforms generated by alternative splicing, alternative promoter usage or alternative initiation of translation, and is commonly mutated in different cancers. Therefore, p53 serves as an important illustration of the protein structure-function continuum concept, where the generation of multiple proteoforms by various mechanisms defines the ability of this protein to have a multitude of structurally and functionally different states. Considering p53 in the light of a proteoform-based structure-function continuum represents a non-canonical and conceptually new contemplation of structure, regulation, and functionality of this important protein.
Topics: Alternative Splicing; Humans; Intrinsically Disordered Proteins; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protein Isoforms; Protein Processing, Post-Translational; Tumor Suppressor Protein p53
PubMed: 27834926
DOI: 10.3390/ijms17111874