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1.
Cell ; 186(18): 3903-3920.e21, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37557169

RESUMO

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.


Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Evasão Tumoral , Humanos , Apresentação de Antígeno , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Neoplasias/imunologia , Ubiquitina-Proteína Ligases/genética
2.
Mol Cell ; 84(7): 1224-1242.e13, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38458201

RESUMO

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.


Assuntos
Ciclinas , Reparo de Erro de Pareamento de DNA , Animais , Ciclinas/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Interfase , Mamíferos/metabolismo
3.
Mol Cell ; 83(1): 1-3, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608666

RESUMO

Cells must avoid licensing of neosynthesized DNA to prevent rereplication. In this issue of Molecular Cell, Ratnayeke et al. (2022)1 reveal how the licensing factor CDT1, prior to its degradation, inhibits DNA elongation by suppressing CMG helicase progression at replication forks.


Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , DNA , DNA Helicases/genética
4.
Nature ; 592(7856): 789-793, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33854235

RESUMO

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Divisão Celular , Ciclina D1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sistemas CRISPR-Cas , Ciclina D2/metabolismo , Ciclina D3/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , Técnicas de Inativação de Genes , Genes Supressores de Tumor , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos , Neoplasias/genética , Ubiquitina/metabolismo
5.
Nature ; 592(7856): 799-803, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33854232

RESUMO

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclina D/metabolismo , Instabilidade Genômica , Fase S , Animais , Linhagem Celular , Proliferação de Células , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Knockout , Mutações Sintéticas Letais
6.
Genes Dev ; 33(23-24): 1615-1616, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31792015

RESUMO

Diverse linkage in polyubiquitin chain structure gives cells an unparalleled complexity to virtually modulate all aspects of cell biology. Substrates can be covalently modified by ubiquitin chains of different topology. Proper DNA damage response takes advantage of this regulatory system and heavily relies on ubiquitin-based signaling. Moreover, increasing evidence suggests that chain specificity dictates DNA repair outcome. In this issue of Genes & Development, Wu and colleagues (pp. 1702-1717) show that Cezanne and Cezanne2, two paralogous deubiquitinating enzymes that are recruited to sites of DNA damage, ensure proper local polyubiquitin chain composition for downstream DNA repair protein assembly. Their study offers a key insight into the mechanism of crosstalk between linkage-specific ubiquitylation at DNA damage sites, while simultaneously raising important questions for future research.


Assuntos
Poliubiquitina , Ubiquitina , Dano ao DNA , Reparo do DNA , Ligação Proteica , Ubiquitinação
7.
Mol Cell ; 64(3): 507-519, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27773672

RESUMO

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.


Assuntos
Ciclinas/genética , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Histonas/genética , Proteínas Nucleares/genética , RNA Mensageiro/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Motivos de Aminoácidos , Animais , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Ciclinas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fosforilação , Polirribossomos/genética , Polirribossomos/metabolismo , Ligação Proteica , Proteólise , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Xenopus laevis , Peixe-Zebra , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
8.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33827988

RESUMO

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Assuntos
COVID-19/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Complexos Multiproteicos/metabolismo , Fases de Leitura Aberta , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , COVID-19/genética , Proteínas de Ciclo Celular/genética , Proteínas Culina/genética , Células HEK293 , Humanos , Complexos Multiproteicos/genética , SARS-CoV-2/genética , Proteínas Virais/genética
9.
Nucleic Acids Res ; 44(3): e28, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26429970

RESUMO

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.


Assuntos
DNA/química , Uracila/análise , Catálise , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro
10.
Nucleic Acids Res ; 42(19): 11912-20, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25274731

RESUMO

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pirofosfatases/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/genética , Proteínas de Bactérias/antagonistas & inibidores , Ilhas Genômicas , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/genética , Proteínas Repressoras/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo
11.
PLoS Genet ; 8(6): e1002738, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22685418

RESUMO

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil-DNA glycosylase and dUTPase. Lack of the major uracil-DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200-2,000 uracil/million bases, quantified using a novel real-time PCR-based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil-DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil-DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil-DNA in this evolutionary clade.


Assuntos
DNA/genética , Drosophila melanogaster/genética , Larva/genética , Pirofosfatases , Uracila , Animais , Linhagem Celular , DNA/química , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto , Instabilidade Genômica , Células HeLa , Humanos , Larva/crescimento & desenvolvimento , Pirofosfatases/genética , Interferência de RNA , Uracila/química , Uracila/metabolismo , Uracila/farmacologia , Uracila-DNA Glicosidase/genética
12.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 10): 2777-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25286861

RESUMO

The authors respond to a comment by Alvisi & Jans [(2014), Acta Cryst. D70, 2775-2776] on the article Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights [Róna et al. (2013), Acta Cryst. D69, 2495-2505].


Assuntos
Pirofosfatases/metabolismo , alfa Carioferinas/metabolismo , Humanos
13.
Res Sq ; 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39070640

RESUMO

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

14.
bioRxiv ; 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38979260

RESUMO

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

15.
bioRxiv ; 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-38260436

RESUMO

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.

16.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 12): 2298-308, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24311572

RESUMO

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Šresolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small ß-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.


Assuntos
Pirofosfatases/química , Pirofosfatases/metabolismo , Fagos de Staphylococcus/enzimologia , Sequência de Aminoácidos , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Alinhamento de Sequência , Fagos de Staphylococcus/química , Staphylococcus aureus/virologia
17.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 12): 2495-505, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24311590

RESUMO

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α-wild-type and the importin-α-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.


Assuntos
Pirofosfatases/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Ciclo Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Sinais de Localização Nuclear , Fosforilação , Pirofosfatases/química , alfa Carioferinas/química
18.
J Vis Exp ; (202)2023 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-38189447

RESUMO

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms. This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.


Assuntos
Reparo do DNA , DNA de Cadeia Simples , Ciclo Celular , Divisão Celular , Fase G1
19.
Sci Adv ; 9(41): eadh1134, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37831778

RESUMO

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.


Assuntos
Endossomos , ATPases Vacuolares Próton-Translocadoras , Autofagia , Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Humanos
20.
Cell Death Differ ; 29(2): 285-292, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34862481

RESUMO

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Exorribonucleases/metabolismo , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células A549 , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Antivirais/farmacologia , Humanos , SARS-CoV-2/enzimologia , Replicação Viral/efeitos dos fármacos
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