RESUMEN
Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.
Asunto(s)
Proteína BRCA2 , Neoplasias de la Mama , Glucólisis , Piruvaldehído , Animales , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Ratones , Humanos , Femenino , Piruvaldehído/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Haploinsuficiencia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Mutación , Daño del ADN , Reparación del ADN , Línea Celular TumoralRESUMEN
Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.
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Proteína BRCA2/genética , Aberraciones Cromosómicas/efectos de los fármacos , Formaldehído/toxicidad , Inestabilidad Genómica/efectos de los fármacos , Toxinas Biológicas/toxicidad , Daño del ADN , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Haploinsuficiencia , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Proteoma , Ribonucleasa H/metabolismoRESUMEN
Genome instability occurs early in the development of most cancers. Bester et al. now provide evidence that oncogenic signals trigger cell division without coordinate nucleotide synthesis, engendering aberrant DNA replication and damage that could promote carcinogenesis. A mismatch between proliferation and metabolite production may characterize oncogenic cell cycles.
RESUMEN
SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.
Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifested by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA-binding selectivity.
Asunto(s)
Proteína BRCA2/metabolismo , ADN de Cadena Simple/metabolismo , Recombinasa Rad51/metabolismo , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Proteína BRCA2/química , Humanos , Modelos Biológicos , Recombinación GenéticaRESUMEN
It is emerging that the pathways that process newly transcribed RNA molecules also regulate the response to DNA damage at multiple levels. Here, we discuss recent insights into how RNA processing pathways participate in DNA damage recognition, signaling, and repair, selectively influence the expression of genome-stabilizing proteins, and resolve deleterious DNA/RNA hybrids (R-loops) formed during transcription and RNA processing. The importance of these pathways for the DNA damage response (DDR) is underscored by the growing appreciation that defects in these regulatory connections may be connected to the genome instability involved in several human diseases, including cancer.
Asunto(s)
Genoma Humano , Inestabilidad Genómica , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Daño del ADN , Reparación del ADN , Humanos , Transducción de SeñalRESUMEN
Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (â¼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.
Asunto(s)
Proteína BRCA2 , Neoplasias de la Mama/genética , Reparación del ADN , Neoplasias Ováricas/genética , Complejo de la Endopetidasa Proteasomal/fisiología , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Femenino , Células HEK293 , Células HeLa , Recombinación Homóloga , Humanos , Mutación Missense , Unión ProteicaRESUMEN
The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.
Asunto(s)
Aurora Quinasa A/fisiología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/metabolismo , Regulación Alostérica , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Puntos de Control de la Fase G1 del Ciclo Celular , Células HeLa , Humanos , Interfase/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Origen de RéplicaRESUMEN
Messenger RNA (mRNA) export from the nucleus is essential for eukaryotic gene expression. Here we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enriched for functions in genome duplication and repair, controlled by inositol polyphosphate multikinase (IPMK), an enzyme catalyzing inositol polyphosphate and phosphoinositide turnover. We studied transcripts encoding RAD51, a protein essential for DNA repair by homologous recombination (HR), to characterize the mechanism underlying IPMK-regulated mRNA export. IPMK depletion or catalytic inactivation selectively decreases RAD51 protein abundance and the nuclear export of RAD51 mRNA, thereby impairing HR. Recognition of a sequence motif in the untranslated region of RAD51 transcripts by the mRNA export factor ALY requires IPMK. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an IPMK product, restores ALY recognition in IPMK-depleted cell extracts, suggesting a mechanism underlying transcript selection. Our findings implicate IPMK in a transcript-selective mRNA export pathway controlled by phosphoinositide turnover that preserves genome integrity in humans.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Inestabilidad Genómica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/genética , Línea Celular Tumoral , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Humano , Recombinación Homóloga/genética , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30-40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut-but do not overlap-with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2.
Asunto(s)
Daño del ADN , Recombinasa Rad51/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Proteína BRCA2/fisiología , Línea Celular , Reparación del ADN , ADN de Cadena Simple , Células HeLa , Humanos , Cinética , Microscopía , Proteína de Replicación A/metabolismoRESUMEN
Two decades of fast-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution with low invasiveness and high specificity. Fluorescence microscopy also enables scientists and clinicians to map and quantitate the physicochemical properties (e.g., analyte concentration, enzymatic activities, and protein-protein interactions) of biological samples. But the biochemical resolving power of fluorescence microscopy is not as well optimized as its spatial resolution. Current techniques typically observe only the individual properties of fluorescence, thus limiting the opportunities for sensing and multiplexing. Here, we demonstrate a new, to our knowledge, imaging paradigm, hyperdimensional imaging microscopy, which quantifies simultaneously and efficiently all the properties of fluorescence emission (excited-state lifetime, polarization, and spectra) in biological samples, transcending existing limitations. Such simultaneous detection of fluorescence features maximizes the biochemical resolving power of fluorescence microscopy, thereby providing the means to enhance sensing capabilities and enable heavily multiplexed assays. Just as multidimensional separation in mass-spectroscopy and multidimensional spectra in NMR have empowered proteomics and structural biology, we envisage that hyperdimensional imaging microscopy spectra of unprecedented dimensionality will catalyze advances in systems biology and medical diagnostics.
Asunto(s)
Microscopía , Proteómica , Biología de SistemasRESUMEN
Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.
Asunto(s)
Microscopía por Crioelectrón , ADN de Cadena Simple/química , Recombinasa Rad51/química , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sitios de Unión , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Predisposición Genética a la Enfermedad , Recombinación Homóloga , Humanos , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Secuencias Repetitivas de AminoácidoRESUMEN
The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.
Asunto(s)
Acetiltransferasas/fisiología , Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Humanos , Proteínas de Transporte Nucleocitoplasmático/fisiología , ARN Mensajero/clasificación , Proteínas de Unión al ARN/fisiología , XenopusRESUMEN
Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.
Asunto(s)
Aumento de la Imagen/instrumentación , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Confocal/instrumentación , Imagen Molecular/instrumentación , Imagen Óptica/instrumentación , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/instrumentación , Lentes , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This inâ situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces inâ situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.
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Compuestos Macrocíclicos/química , Péptidos/química , Medios de Cultivo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/químicaRESUMEN
We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double-click stapling of p53-based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53-activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.
Asunto(s)
Alquinos/química , Antineoplásicos/química , Péptidos/química , Proteína p53 Supresora de Tumor/química , Alquinos/farmacología , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Azidas/química , Azidas/farmacología , Línea Celular , Química Clic , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Stapling peptides for inhibiting the p53/MDM2 interaction is a promising strategy for developing anti-cancer therapeutic leads. We evaluate double-click stapled peptides formed from p53-based diazidopeptides with different staple positions and azido amino acid side-chain lengths, determining the impact of these variations on MDM2 binding and cellular activity. We also demonstrate a K24R mutation, necessary for cellular activity in hydrocarbon-stapled p53 peptides, is not required for analogous 'double-click' peptides.
Asunto(s)
Química Clic/métodos , Péptidos/química , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Dicroismo Circular , Polarización de Fluorescencia , Genes Reporteros , Datos de Secuencia MolecularRESUMEN
Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Fibroblastos , Histonas/metabolismo , Humanos , Enlace de Hidrógeno , Metilación , Ratones , Mutación , Fosforilación , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1-containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Catálisis , Ciclo Celular , Centrosoma/ultraestructura , Citoplasma/metabolismo , Difusión , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Sustancias Macromoleculares , Microscopía Confocal/métodos , Mutación Puntual , Epitelio Pigmentado de la Retina/citología , Programas Informáticos , Espectrofotometría/métodos , Quinasa Tipo Polo 1RESUMEN
Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.