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Methods in Molecular Biology (Clifton,... 2023Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads...
Cellular division is a fundamental process of cellular growth. First, cells replicate their DNA in S phase and then undergo mitosis which, under normal conditions, leads to complete cell division. Moreover, mitotic activity correlates to cellular growth activity. The simplest and classical method to measure mitotic activity (mitotic index (MI)), is the manual counting of mitotic cells among a given cell population of interest. The latter can be accomplished via phase contrast microscope observation. However, Giemsa staining may improve accuracy and consistency. Fluorescence immunostaining targeting specific phosphorylations of proteins at critical cell cycle steps will provide further improved analysis via high-throughput capacity of flow or imaging cytometer. Finally, time lapse image analysis provides quantitative and qualitative metrics delineating the process of cellular division including timing of division, duration of mitosis, and failure to procced through or complete mitosis.
Topics: Cell Cycle; Mitosis; Mitotic Index; Phosphorylation; S Phase
PubMed: 36066706
DOI: 10.1007/978-1-0716-2433-3_3 -
Chembiochem : a European Journal of... Jan 2021Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically... (Review)
Review
Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphoproteome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies.
Topics: Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Proteome
PubMed: 33094900
DOI: 10.1002/cbic.202000583 -
Science Advances Apr 2022Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal,...
Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.
Topics: DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Phosphorylation
PubMed: 35353580
DOI: 10.1126/sciadv.abm0314 -
Journal of the American Chemical Society Aug 2020Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via...
Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases-AMPK and PKC-to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton's tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.
Topics: Molecular Structure; Phosphorylation; Small Molecule Libraries
PubMed: 32787262
DOI: 10.1021/jacs.0c05537 -
Journal of Proteome Research Jun 2023Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of...
Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of specific regulating actions of enzymes, such as tyrosine kinases phosphorylating tyrosine residues or by nonenzymatic reactions, such as oxidation related to oxidative stress and diseases. While many studies have addressed the multisite, dynamic, and network-like properties of PTMs, only little is known of the interplay of the same site modifications. In this work, we studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which tyrosine residues were replaced with l-DOPA. The phosphorylated peptides were identified by liquid chromatography-high-resolution mass spectrometry and the site of phosphorylation by tandem mass spectrometry. The results clearly show that the oxidized tyrosine residues are phosphorylated, displaying a specific immonium ion peak in the MS spectra. Furthermore, we detected this modification in our reanalysis (MassIVE ID: MSV000090106) of published bottom-up phosphoproteomics data. The modification, where both oxidation and phosphorylation take place at the same amino acid, has not yet been published in PTM databases. Our data indicate that there can be multiple PTMs that do not exclude each other at the same modification site.
Topics: Phosphorylation; Tyrosine; Levodopa; Peptides; Tandem Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 37146082
DOI: 10.1021/acs.jproteome.3c00061 -
Frontiers in Immunology 2022The SARS-CoV-2 infection triggers host kinases and is responsible for heavy phosphorylation in the host and also in the virus. Notably, phosphorylations in virus were... (Review)
Review
The SARS-CoV-2 infection triggers host kinases and is responsible for heavy phosphorylation in the host and also in the virus. Notably, phosphorylations in virus were achieved using the host enzyme for its better survival and further mutations. We have attempted to study and understand the changes that happened in phosphorylation during and post SARS-CoV-2 infection. There were about 70 phosphorylation sites detected in SARS-CoV-2 viral proteins including N, M, S, 3a, and 9b. Furthermore, more than 15,000 host phosphorylation sites were observed in SARS-CoV-2-infected cells. SARS-CoV-2 affects several kinases including CMGC, CK2, CDK, PKC, PIKFYVE, and EIF2AK2. Furthermore, SARS-CoV-2 regulates various signaling pathways including MAPK, GFR signaling, TGF-β, autophagy, and AKT. These elevated kinases and signaling pathways can be potential therapeutic targets for anti-COVID-19 drug discovery. Specific inhibitors of these kinases and interconnected signaling proteins have great potential to cure COVID-19 patients and slow down the ongoing COVID-19 pandemic.
Topics: Antiviral Agents; Autophagy; Humans; Phosphorylation; Signal Transduction; COVID-19 Drug Treatment
PubMed: 35251015
DOI: 10.3389/fimmu.2022.829474 -
International Journal of Molecular... Jun 2022α-Synuclein is a protein with a molecular weight of 14.5 kDa and consists of 140 amino acids encoded by the gene. Missense mutations and gene duplications in the gene... (Review)
Review
α-Synuclein is a protein with a molecular weight of 14.5 kDa and consists of 140 amino acids encoded by the gene. Missense mutations and gene duplications in the gene cause hereditary Parkinson's disease. Highly phosphorylated and abnormally aggregated α-synuclein is a major component of Lewy bodies found in neuronal cells of patients with sporadic Parkinson's disease, dementia with Lewy bodies, and glial cytoplasmic inclusion bodies in oligodendrocytes with multiple system atrophy. Aggregated α-synuclein is cytotoxic and plays a central role in the pathogenesis of the above-mentioned synucleinopathies. In a healthy brain, most α-synuclein is unphosphorylated; however, more than 90% of abnormally aggregated α-synuclein in Lewy bodies of patients with Parkinson's disease is phosphorylated at Ser129, which is presumed to be of pathological significance. Several kinases catalyze Ser129 phosphorylation, but the role of phosphorylation enzymes in disease pathogenesis and their relationship to cellular toxicity from phosphorylation are not fully understood in α-synucleinopathy. Consequently, this review focuses on the pathogenic impact of α-synuclein phosphorylation and its kinases during the neurodegeneration process in α-synucleinopathy.
Topics: Humans; Lewy Bodies; Parkinson Disease; Phosphorylation; Synucleinopathies; alpha-Synuclein
PubMed: 35682892
DOI: 10.3390/ijms23116216 -
EMBO Reports Sep 2015Ubiquitylation is among the most prevalent post-translational modifications (PTMs) and regulates numerous cellular functions. Interestingly, ubiquitin (Ub) can be itself... (Review)
Review
Ubiquitylation is among the most prevalent post-translational modifications (PTMs) and regulates numerous cellular functions. Interestingly, ubiquitin (Ub) can be itself modified by other PTMs, including acetylation and phosphorylation. Acetylation of Ub on K6 and K48 represses the formation and elongation of Ub chains. Phosphorylation of Ub happens on multiple sites, S57 and S65 being the most frequently modified in yeast and mammalian cells, respectively. In mammals, the PINK1 kinase activates ubiquitin ligase Parkin by phosphorylating S65 of Ub and of the Parkin Ubl domain, which in turn promotes the amplification of autophagy signals necessary for the removal of damaged mitochondria. Similarly, TBK1 phosphorylates the autophagy receptors OPTN and p62 to initiate feedback and feedforward programs for Ub-dependent removal of protein aggregates, mitochondria and pathogens (such as Salmonella and Mycobacterium tuberculosis). The impact of PINK1-mediated phosphorylation of Ub and TBK1-dependent phosphorylation of autophagy receptors (OPTN and p62) has been recently linked to the development of Parkinson's disease and amyotrophic lateral sclerosis, respectively. Hence, the post-translational modification of Ub and its receptors can efficiently expand the Ub code and modulate its functions in health and disease.
Topics: Acetylation; Amyotrophic Lateral Sclerosis; Animals; Autophagy; Humans; Mitophagy; Parkinson Disease; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Protein Structure, Tertiary; Ubiquitin; Ubiquitination; Yeasts
PubMed: 26268526
DOI: 10.15252/embr.201540891 -
Trends in Biochemical Sciences Mar 2022Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein... (Review)
Review
Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/β-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing β-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of β-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy.
Topics: COVID-19; Cell Adhesion; Humans; Integrins; Phosphorylation; SARS-CoV-2
PubMed: 34872819
DOI: 10.1016/j.tibs.2021.11.003 -
International Journal of Molecular... Jun 2022Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes.... (Review)
Review
Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1-2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.
Topics: Animals; Biological Phenomena; Hormones; Phosphorylation; Plant Development; Plant Growth Regulators; Plants; Protein Processing, Post-Translational; Serine; Threonine; Tyrosine
PubMed: 35743047
DOI: 10.3390/ijms23126603