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Molecular & Cellular Proteomics : MCP Dec 2013Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective...
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.
Topics: Carrier Proteins; Cell Line, Tumor; Chromatography, Gel; Databases, Protein; Gene Expression Regulation; Genetic Variation; Histone Acetyltransferases; Humans; Internet; Intracellular Signaling Peptides and Proteins; Mass Spectrometry; Nuclear Proteins; Osteoblasts; Phosphorylation; Protein Binding; Protein Isoforms; Protein Multimerization; Protein Processing, Post-Translational; Proteome; Proteomics; Reproducibility of Results; Solubility; Transcription Factors
PubMed: 24043423
DOI: 10.1074/mcp.M113.032367 -
Biochemical Society Transactions Jun 2023The 3' untranslated region (3'UTR) of mRNA plays a key role in the post-transcriptional regulation of gene expression. Most eukaryotic protein-coding genes express 3'UTR... (Review)
Review
The 3' untranslated region (3'UTR) of mRNA plays a key role in the post-transcriptional regulation of gene expression. Most eukaryotic protein-coding genes express 3'UTR isoforms owing to alternative cleavage and polyadenylation (APA). The 3'UTR isoform expression profile of a cell changes in cell proliferation, differentiation, and stress conditions. Here, we review the emerging theme of regulation of 3'UTR isoforms in cell metabolic reprogramming, focusing on cell growth and autophagy responses through the mTOR pathway. We discuss regulatory events that converge on the Cleavage Factor I complex, a master regulator of APA in 3'UTRs, and recent understandings of isoform-specific m6A modification and endomembrane association in determining differential metabolic fates of 3'UTR isoforms.
Topics: 3' Untranslated Regions; RNA Isoforms; Gene Expression Regulation; Polyadenylation; Protein Isoforms
PubMed: 37171086
DOI: 10.1042/BST20221128 -
Clinical Epigenetics 2016DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our... (Review)
Review
DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our environment, and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases, provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (<10 %) changes to intermediate methylation levels are associated with complex disease phenotypes. This has resulted in two clear methylation paradigms. The latter "subtle change" paradigm is rapidly becoming the epigenetic hallmark of complex disease phenotypes, although we are currently hampered by a lack of data addressing the true biological significance and meaning of these small differences. Our initial expectation of rapidly identifying mechanisms linking environmental exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5'UTR splicing, and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity, providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in the NR3C1 CpG island alters the NR3C1 transcription and eventually protein isoforms in the tissues, resulting in subtle but visible physiological variability. This review addresses the current pathophysiological and clinical associations of such characteristically small DNA methylation changes, the ever-growing roles of DNA methylation and the evidence available, particularly from the glucocorticoid receptor of the cascade of events initiated by such subtle methylation changes, as well as addressing the underlying question as to what represents a genuine biologically significant difference in methylation.
Topics: 5' Untranslated Regions; 5-Methylcytosine; CpG Islands; DNA Methylation; Environmental Exposure; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation; Humans; Phenotype; Protein Isoforms; Receptors, Glucocorticoid
PubMed: 27602172
DOI: 10.1186/s13148-016-0256-8 -
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 -
Physiological Reviews Jan 2000Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status... (Review)
Review
Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na(+)-K(+)-Cl(-) cotransporter (NKCC) protein (approximately 120-130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly all cell types. The NKCC maintains intracellular Cl(-) concentration ([Cl(-)](i)) at levels above the predicted electrochemical equilibrium. The high [Cl(-)](i) is used by epithelial tissues to promote net salt transport and by neural cells to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions. Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with evidence for the electrically neutral stoichiometries of 1 Na(+):1 K(+):2 Cl- (for most cells) and 2 Na(+):1 K(+):3 Cl(-) (in squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been identified. An unusual form of NKCC regulation is by [Cl(-)](i). [Cl(-)](i) in the physiological range and above strongly inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism.
Topics: Animals; Carrier Proteins; Cell Membrane; Chlorides; Humans; Kidney; Potassium; Protein Isoforms; Sodium; Sodium-Potassium-Chloride Symporters
PubMed: 10617769
DOI: 10.1152/physrev.2000.80.1.211 -
Biological Chemistry May 2017The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid... (Review)
Review
The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.
Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Membrane; Humans; Lipid Metabolism; Membranes, Artificial; Protein Isoforms; ras Proteins
PubMed: 27977396
DOI: 10.1515/hsz-2016-0289 -
Seminars in Cell & Developmental Biology Oct 2016Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in... (Review)
Review
Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.
Topics: Amino Acid Sequence; Animals; Humans; Models, Biological; Mutation; Neoplasms; Protein Isoforms; Signal Transduction; ras Proteins
PubMed: 27422332
DOI: 10.1016/j.semcdb.2016.07.012 -
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 -
Cell Cycle (Georgetown, Tex.) Dec 2012p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the... (Review)
Review
p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the N-terminal transactivation domain (TA isoform) and one N-terminal truncated isoform (ΔN isoform). Complete ablation of all p63 isoforms produced mice with fatal developmental abnormalities, including lack of epidermal barrier, limbs and other epidermal appendages. Specific TAp63-null mice, although they developed normally, failed to undergo in DNA damage-induced apoptosis during primordial follicle meiotic arrest, suggesting a p63 involvement in maternal reproduction. Recent findings have elucidated the role in DNA damage response of a novel Hominidae p63 isoform, GTAp63, specifically expressed in human spermatic precursors. Thus, these findings suggest a unique strategy of p63 gene, to evolve in order to preserve the species as a guardian of reproduction. Elucidation of the biological basis of p63 function in reproduction may provide novel approaches to the control of human fertility.
Topics: Animals; Female; Fertility; Humans; Mice; Protein Isoforms; Transcription Factors; Tumor Suppressor Proteins
PubMed: 23165243
DOI: 10.4161/cc.22819 -
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