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1.
Mol Cell ; 83(5): 770-786.e9, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36805027

RESUMO

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.


Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Transporte , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
J Biol Chem ; 299(12): 105427, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37926283

RESUMO

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates versus complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase. This pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP toward DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.


Assuntos
Neoplasias , Separação de Fases , Humanos , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais
3.
Mol Cell ; 59(2): 176-87, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26145171

RESUMO

The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.


Assuntos
Proteína BRCA2/metabolismo , Recombinação Homóloga , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína de Replicação A/metabolismo , Substituição de Aminoácidos , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular , Feminino , Células HeLa , Humanos , Modelos Biológicos , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/genética
4.
Proc Natl Acad Sci U S A ; 113(12): 3275-80, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26951671

RESUMO

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY-ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA-gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA-UvsX filament.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Proteínas Virais/química , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-Atividade
5.
J Biol Chem ; 287(9): 6469-81, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22194613

RESUMO

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.


Assuntos
Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteína de Replicação A/metabolismo , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Cromossomos/fisiologia , DNA Helicases/química , DNA Helicases/genética , Células HCT116 , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Osteossarcoma , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteína de Replicação A/química , Proteína de Replicação A/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia
6.
Proc Natl Acad Sci U S A ; 107(31): 13684-9, 2010 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-20643958

RESUMO

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.


Assuntos
DNA Primase/química , Primers do DNA/química , Ferro/química , Domínios e Motivos de Interação entre Proteínas , Enxofre/química , Cristalografia por Raios X , DNA Primase/metabolismo , Primers do DNA/metabolismo , Humanos , Ferro/metabolismo , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Enxofre/metabolismo
7.
bioRxiv ; 2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36993550

RESUMO

Phase separation is a ubiquitous process that compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of complexes below the saturation concentration, the contribution of condensates vs complexes to function is not always clear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3), which pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to the substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX, but enhance the poly-ubiquitination activity of SPOP towards DAXX. This unexpectedly enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of clusters versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate its activity, and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of these cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

8.
Eur J Med Chem ; 247: 115035, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36603507

RESUMO

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.


Assuntos
Dibenzotiepinas , Inibidores de Integrase de HIV , Influenza Humana , Orthomyxoviridae , Humanos , RNA Polimerase Dependente de RNA , Piridonas/farmacologia , Piridonas/uso terapêutico , Influenza Humana/tratamento farmacológico , Dibenzotiepinas/farmacologia , Dibenzotiepinas/uso terapêutico , Endonucleases , Triazinas/farmacologia , Antivirais/farmacologia
9.
Arch Biochem Biophys ; 518(2): 111-8, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22227098

RESUMO

Simian Virus 40 uses the large T antigen (Tag) to bind and inactivate retinoblastoma tumor suppressor proteins (Rb), which can result in cellular transformation. Tag is a modular protein with four domains connected by flexible linkers. The N-terminal J domain of Tag is necessary for Rb inactivation. Binding of Rb is mediated by an LXCXE consensus motif immediately C-terminal to the J domain. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) were used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N(260)) extending from the J domain through the origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the domains in N(260). Binding of pRb to a construct containing the LXCXE motif and the J domain revealed weak interactions between pRb and the J domain. Analysis of the complex of pRb and N(260) indicated that the OBD is not involved and retains its dynamic independence from the remainder of Tag. These results support a 'chaperone' model in which the J domain of Tag changes its orientation as it acts upon different protein complexes.


Assuntos
Antígenos Transformantes de Poliomavirus/química , Complexos Multiproteicos/química , Proteína do Retinoblastoma/química , Motivos de Aminoácidos , Antígenos Transformantes de Poliomavirus/metabolismo , Complexos Multiproteicos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína do Retinoblastoma/metabolismo
10.
Nat Commun ; 13(1): 250, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017502

RESUMO

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.


Assuntos
Apoptose/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Morte Celular , Cristalografia por Raios X , Humanos , Ligantes , Lipossomos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química
11.
Sci Transl Med ; 14(653): eabq2096, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35857643

RESUMO

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.


Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Carcinogênese/genética , Linhagem Celular Tumoral , Criança , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/uso terapêutico , Rabdomiossarcoma/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia
12.
iScience ; 24(1): 101996, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33490904

RESUMO

Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.

13.
Structure ; 16(12): 1892-901, 2008 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-19081065

RESUMO

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/química , DNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Biofísicos , DNA/genética , Análise Mutacional de DNA , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Proteínas de Manutenção de Minicromossomo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Dedos de Zinco/genética
14.
Nat Commun ; 10(1): 1676, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976006

RESUMO

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.


Assuntos
Divisão Celular/fisiologia , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Cristalografia por Raios X , Ciclina A/isolamento & purificação , Quinase 2 Dependente de Ciclina/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/isolamento & purificação , Proteínas de Fusão bcr-abl/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo , Tirosina/metabolismo , Quinases da Família src/isolamento & purificação , Quinases da Família src/metabolismo
15.
J Mol Biol ; 430(17): 2857-2872, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29864443

RESUMO

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Éteres Fenílicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina/química , Ubiquitina/metabolismo , Sequência de Aminoácidos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Cromatina/química , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Dano ao DNA , Humanos , Modelos Moleculares , Proteínas Nucleares/efeitos dos fármacos , Nucleotidiltransferases/efeitos dos fármacos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Células Tumorais Cultivadas , Ubiquitina/efeitos dos fármacos , Ubiquitinação
16.
J Mol Biol ; 426(3): 558-69, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24239947

RESUMO

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8-10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn(2+) but not Mg(2+) significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn(2+) provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.


Assuntos
DNA Primase/química , Primers do DNA/síntese química , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA Primase/metabolismo , Humanos , Manganês/metabolismo , Conformação Proteica , Subunidades Proteicas , Saccharomyces cerevisiae/metabolismo
17.
Mol Cell Biol ; 31(21): 4298-309, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21859891

RESUMO

Proapoptotic BH3-interacting death domain agonist (BID) regulates apoptosis and the DNA damage response. Following replicative stress, BID associates with proteins of the DNA damage sensor complex, including replication protein A (RPA), ataxia telangiectasia and Rad3 related (ATR), and ATR-interacting protein (ATRIP), and facilitates an efficient DNA damage response. We have found that BID stimulates the association of RPA with components of the DNA damage sensor complex through interaction with the basic cleft of the N-terminal domain of the RPA70 subunit. Disruption of the BID-RPA interaction impairs the association of ATR-ATRIP with chromatin as well as ATR function, as measured by CHK1 activation and recovery of DNA replication following hydroxyurea (HU). We further demonstrate that the association of BID with RPA stimulates the association of ATR-ATRIP to the DNA damage sensor complex. We propose a model in which BID associates with RPA and stimulates the recruitment and/or stabilization of ATR-ATRIP to the DNA damage sensor complex.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Sequência de Bases , Proteínas de Ciclo Celular/química , Linhagem Celular , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/química , Transdução de Sinais , Estresse Fisiológico
18.
Mol Cell Biol ; 28(24): 7345-53, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18936170

RESUMO

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína de Replicação A/genética , Alinhamento de Sequência
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