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1.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639033

RESUMO

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.


Assuntos
Acetiltransferase N-Terminal A/química , Acetiltransferase N-Terminal A/metabolismo , Acetilação , Catálise , Quinases Ciclina-Dependentes/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Metabolismo dos Lipídeos , Acetiltransferase N-Terminal A/genética , Proteoma , Proteômica/métodos , Especificidade por Substrato
2.
Nat Commun ; 12(1): 6053, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663829

RESUMO

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/ .


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteoma/metabolismo , Transdução de Sinais , Células A549 , Apoptose , Morte Celular , Linhagem Celular , Citocinas/metabolismo , Humanos , Necroptose , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Células U937
3.
Life Sci ; 285: 120006, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34606852

RESUMO

Neurodegenerative diseases (NDDs) are the most common life-threatening disease of the central nervous system and it cause the progressive loss of neuronal cells. The exact mechanism of the disease's progression is not clear and thus line of treatment for NDDs is a baffling issue. During the progression of NDDs, oxidative stress and DNA damage play an important regulatory function, and ultimately induces neurodegeneration. Recently, aberrant cell cycle events have been demonstrated in the progression of different NDDs. However, the pertinent role of signaling mechanism, for instance, post-translational modifications, oxidative stress, DNA damage response pathway, JNK/p38 MAPK, MEK/ERK cascade, actively participated in the aberrant cell cycle reentry induced neuronal cell death. Mounting evidence has demonstrated that aberrant cell cycle re-entry is a major contributing factor in the pathogenesis of NDDs rather than a secondary phenomenon. In the brain of AD patients with mild cognitive impairment, post miotic cell division can be seen in the early stage of the disease. However, in the brain of PD patients, response to various neurotoxic signals, the cell cycle re-entry has been observed that causes neuronal apoptosis. On contrary, the contributing factors that leads to the induction of cell cycle events in mature neurons in HD and ALS brain pathology is remain unclear. Various pharmacological drugs have been developed to reduce the pathogenesis of NDDs, but they are still not helpful in eliminating the cause of these NDDs.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Mitose , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Acetilação , Animais , Histona Desacetilases/metabolismo , Humanos , Ubiquitina-Proteína Ligases/metabolismo
4.
Nat Commun ; 12(1): 5224, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471130

RESUMO

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição
5.
J Biol Chem ; 297(4): 101162, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481843

RESUMO

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-ß) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-ß family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-ß/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-ß/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Subunidades beta de Inibinas/metabolismo , Proteínas de Neoplasias/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação para Cima/efeitos dos fármacos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Subunidades beta de Inibinas/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
6.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586062

RESUMO

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.


Assuntos
Meiose , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
J Neurosci ; 41(40): 8309-8320, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34429379

RESUMO

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF-CDC42-MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration.SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14-EPHX-1-CDC-42-MLC phosphorylation axis.


Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Regeneração Nervosa/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Chlorocebus aethiops , Quinases Ciclina-Dependentes/genética
8.
Cell Mol Life Sci ; 78(14): 5543-5567, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34146121

RESUMO

Cyclin-dependent kinase 9 (CDK9), the kinase component of positive transcription elongation factor b (P-TEFb), is essential for transcription of most protein-coding genes by RNA polymerase II (RNAPII). By releasing promoter-proximally paused RNAPII into gene bodies, CDK9 controls the entry of RNAPII into productive elongation and is, therefore, critical for efficient synthesis of full-length messenger (m)RNAs. In recent years, new players involved in P-TEFb-dependent processes have been identified and an important function of CDK9 in coordinating elongation with transcription initiation and termination has been unveiled. As the regulatory functions of CDK9 in gene expression continue to expand, a number of human pathologies, including cancers, have been associated with aberrant CDK9 activity, underscoring the need to properly regulate CDK9. Here, I provide an overview of CDK9 function and regulation, with an emphasis on CDK9 dysregulation in human diseases.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Animais , Quinase 9 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Humanos , Fosforilação , RNA Polimerase II/genética
9.
Methods Mol Biol ; 2329: 265-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085229

RESUMO

The cell cycle is an ordered series of events by which cells grow and divide to give rise to two daughter cells. In eukaryotes, cyclin-cyclin-dependent kinase (cyclin-Cdk) complexes act as master regulators of the cell division cycle by phosphorylating numerous substrates. Their activity and expression profiles are regulated in time. The budding yeast S. cerevisiae was one of the pioneering model organisms to study the cell cycle. Its genetic amenability continues to make it a favorite model to decipher the principles of how changes in cyclin-Cdk activity translate into the intricate sequence of substrate phosphorylation events that govern the cell cycle. In this chapter, we introduce robust and straightforward methods to analyze cell cycle progression in S. cerevisiae. These techniques can be utilized to describe cell cycle events and to address the effects of perturbations on accurate and timely cell cycle progression.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Saccharomyces cerevisiae/fisiologia , Ciclo Celular , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Modelos Biológicos , Feromônios/farmacologia , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065149

RESUMO

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21Waf1/Cip1, p27Kip1, or p16Ink4a. c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin's anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Tricodermina/farmacologia , Animais , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias Ovarianas , Tricodermina/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072267

RESUMO

The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell-cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), the activities of which are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins and CDK inhibitors (CKIs). Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell-cycle process via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor proteins and oncoproteins or, inversely, abnormally high accumulation results in cell proliferation deregulation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the UPS machinery regulation of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell-cycle transition, cancer treatment, and the development of anti-cancer drugs.


Assuntos
Ciclo Celular , Neoplasias/metabolismo , Animais , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Progressão da Doença , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Neoplasias/etiologia , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
DNA Repair (Amst) ; 105: 103143, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34144487

RESUMO

The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.


Assuntos
Antineoplásicos/farmacologia , Reparo do DNA , Neoplasias/metabolismo , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/metabolismo , Antineoplásicos/uso terapêutico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
13.
Mol Cell Biol ; 41(8): e0023421, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34096775

RESUMO

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Fosforilação/fisiologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Espectrometria de Massas em Tandem/métodos , Ubiquitinação/fisiologia
14.
Elife ; 102021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34114564

RESUMO

Maintenance of cell size homeostasis is a property that is conserved throughout eukaryotes. Cell size homeostasis is brought about by the co-ordination of cell division with cell growth and requires restriction of smaller cells from undergoing mitosis and cell division, whilst allowing larger cells to do so. Cyclin-CDK is the fundamental driver of mitosis and therefore ultimately ensures size homeostasis. Here we dissect determinants of CDK activity in vivo to investigate how cell size information is processed by the cell cycle network in fission yeast. We develop a high-throughput single-cell assay system of CDK activity in vivo and show that inhibitory tyrosine phosphorylation of CDK encodes cell size information, with the phosphatase PP2A aiding to set a size threshold for division. CDK inhibitory phosphorylation works synergistically with PP2A to prevent mitosis in smaller cells. Finally, we find that diploid cells of equivalent size to haploid cells exhibit lower CDK activity in response to equal cyclin-CDK enzyme concentrations, suggesting that CDK activity is reduced by increased DNA levels. Therefore, scaling of cyclin-CDK levels with cell size, CDK inhibitory phosphorylation, PP2A, and DNA-dependent inhibition of CDK activity, all inform the cell cycle network of cell size, thus contributing to cell size homeostasis.


Assuntos
Divisão Celular , Tamanho Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ciclo Celular , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Homeostase , Mitose , Fosforilação , Ploidias , Proteína Fosfatase 2/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
15.
PLoS Genet ; 17(6): e1009601, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34086674

RESUMO

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histonas/genética , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Evolução Molecular , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilação , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transgenes
16.
Methods Mol Biol ; 2329: 1-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085211

RESUMO

The cell cycle is the sequence of events through which a cell duplicates its genome, grows, and divides. Key cell cycle transitions are driven by oscillators comprising of protein kinases and their regulators. Different cell cycle oscillators are inextricably linked to ensure orderly activation of oscillators. A recurring theme in their regulation is the abundance of autoamplifying loops that ensure switch-like and unidirectional cell cycle transitions. The periodicity of many cell cycle oscillators is choreographed by inherent mechanisms that promote automatic inactivation, often involving dephosphorylation and ubiquitin-mediated protein degradation. These inhibitory signals are subsequently suppressed to enable the next cell cycle to occur. Although the activation and inactivation of cell cycle oscillators are in essence autonomous during the unperturbed cell cycle, a number of checkpoint mechanisms are able to halt the cell cycle until preconditions or defects are addressed. Together, these mechanisms orchestrate orderly progression of the cell cycle to produce more cells and to safeguard genome stability.


Assuntos
Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Instabilidade Genômica , Animais , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Fosforilação , Proteólise , Transdução de Sinais
17.
Methods Mol Biol ; 2329: 95-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085218

RESUMO

Protein kinases are common elements in multiple signaling networks, influencing numerous downstream processes by directly phosphorylating specific target proteins. During the cell cycle, multiple complexes, each comprising one cyclin and one cyclin-dependent kinase (Cdk), function to regulate the orderly progression of cell cycle events. The mechanisms of cyclin-Cdk mediated control have, in part, been established through biochemical experiments involving the purification of cyclin and Cdk proteins to evaluate the activity of a given complex toward its target substrate proteins.Here I present a detailed procedure to simplify the preparation of cyclin-Cdk complexes by purifying them as a single fusion molecule with a 1:1 molar ratio and a detailed protocol for performing reconstituted kinases assays with the purified complexes.This methodology has allowed us to measure the activity and specificity of all budding yeast cyclin-Cdk1 complexes toward the model substrate histone H1. In addition, it has allowed us to perform kinase assays with a panel of purified human cyclin-Cdk complexes to analyze their specificity toward the retinoblastoma protein (Rb) and map the substrate cyclin-Cdk kinase docking interactions between Rb and human G1-Cdk complex.This chapter is focused on purification of cell cycle cyclin-Cdk complexes, but also affords a generalizable framework that can be adapted to other cyclin-dependent kinases like transcriptional cyclin-Cdks or any other multisubunit enzyme complexes. Taken together, the described workflow is a powerful and flexible biochemical platform for solving long-standing biological questions and has potential value in synthetic biology and in therapeutic discovery.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/isolamento & purificação , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Ciclo Celular , Cromatografia de Afinidade , Quinases Ciclina-Dependentes/química , Humanos , Simulação de Acoplamento Molecular , Complexos Multiproteicos/química , Fosforilação , Ligação Proteica
18.
Sci Rep ; 11(1): 11659, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079040

RESUMO

As like in mammalian system, the DNA damage responsive cell cycle checkpoint functions play crucial role for maintenance of genome stability in plants through repairing of damages in DNA and induction of programmed cell death or endoreduplication by extensive regulation of progression of cell cycle. ATM and ATR (ATAXIA-TELANGIECTASIA-MUTATED and -RAD3-RELATED) function as sensor kinases and play key role in the transmission of DNA damage signals to the downstream components of cell cycle regulatory network. The plant-specific NAC domain family transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) plays crucial role in transducing signals from both ATM and ATR in presence of double strand breaks (DSBs) in the genome and found to play crucial role in the regulation of key genes involved in cell cycle progression, DNA damage repair, endoreduplication and programmed cell death. Here we report that Arabidopsis exposed to high salinity shows generation of oxidative stress induced DSBs along with the concomitant induction of endoreduplication, displaying increased cell size and DNA ploidy level without any change in chromosome number. These responses were significantly prominent in SOG1 overexpression line than wild-type Arabidopsis, while sog1 mutant lines showed much compromised induction of endoreduplication under salinity stress. We have found that both ATM-SOG1 and ATR-SOG1 pathways are involved in the salinity mediated induction of endoreduplication. SOG1was found to promote G2-M phase arrest in Arabidopsis under salinity stress by downregulating the expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1, and CYCB1;1, while upregulating the expression of WEE1 kinase, CCS52A and E2Fa, which act as important regulators for induction of endoreduplication. Our results suggest that Arabidopsis undergoes endoreduplicative cycle in response to salinity induced DSBs, showcasing an adaptive response in plants under salinity stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , DNA de Plantas/genética , Endorreduplicação , Tolerância ao Sal/genética , Fatores de Transcrição/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Tamanho Celular , Ciclina B/genética , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA de Plantas/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Poliploidia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Salino/genética , Transdução de Sinais , Cloreto de Sódio/farmacologia , Fatores de Transcrição/metabolismo
19.
Eur J Med Chem ; 221: 113481, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33945934

RESUMO

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.


Assuntos
Anilidas/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Anilidas/síntese química , Anilidas/química , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
20.
Science ; 372(6546)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33958484

RESUMO

The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.


Assuntos
Complexo Mediador/química , RNA Polimerase II/química , Fator de Transcrição TFIID/química , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Humanos , Complexo Mediador/metabolismo , Subunidade 1 do Complexo Mediador/química , Modelos Moleculares , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , RNA Polimerase II/metabolismo , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo
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