RESUMO
DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA , Morte Celular , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismoRESUMO
REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal ß-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.
Assuntos
Proteínas de Bactérias/metabolismo , Guanosina Difosfato/metabolismo , Proteínas Mad2/química , Proteínas Mad2/metabolismo , Shigella/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Análise Mutacional de DNA , Guanosina Trifosfato/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Proteína ran de Ligação ao GTP/químicaRESUMO
MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a 'closed' active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an 'open' inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus ß-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2's C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2's preferential complex formation.
Assuntos
Antígenos CD/metabolismo , Proteínas Cdh1/metabolismo , Proteínas Mad2/metabolismo , Antígenos CD/genética , Sítios de Ligação , Proteínas Cdh1/genética , Células HEK293 , Humanos , Proteínas Mad2/genética , Nucleotidiltransferases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
OBJECTIVES: SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1. METHODS: SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation. RESULTS: SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1. CONCLUSIONS: Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.
Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Acetilação , MetilaçãoRESUMO
Purpose: We aimed to examine how various types of cancer, classified histologically, affect semen quality. Methods: The study group included 313 patients who were diagnosed with cancer and reached for a sperm cryopreservation before a gonadotoxic treatment (PG-Tx group). Their semen parameters were compared to those of two control groups: (a) individuals who attended a fertility investigation and were found to be above the limit of the lower reference value of the WHO 2010 manual (ARL group), and (b) fertile men, whose semen parameters were obtained from the dataset of the WHO 2020 manual. Results: Semen quality was significantly poorer in the PG-Tx group than in the ARL group. Differences included a 65.6% decrease in concentration, a 12.1% decrease in volume, a 72.7% decrease in total count, and a 33.0%, 22.2%, and 24.7% decrease in total motility, rapid motility, and progressive motility, respectively. Linear regression models comparing the PG-Tx and ARL groups revealed that the maximum reduction in total motility and concentration was in men with germ-cell tumors, whereas the minimum reduction was in hematological tumors. Similarly, all sperm quality parameters were significantly lower in the PG-Tx group than in the fertile-men group (p < 0.0001). Conclusions: While the effect of malignancy on semen parameters is debatable, we found that all examined types of cancer significantly impaired sperm quality parameters. Although the median of most semen parameters of patients with cancer were still in the normal WHO range, their fifth percentile, represents men with a delayed time to pregnancy.
RESUMO
Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.
Assuntos
Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Leishmania donovani/genética , Ciclossomo-Complexo Promotor de Anáfase , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Genoma de Protozoário , Leishmania donovani/citologia , Leishmania donovani/metabolismo , Filogenia , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
BACKGROUND/AIM: Germline mutations in PTCH1 or SUFU in the sonic hedgehog (SHH) pathway cause Gorlin's syndrome with increased risk of developing SHH-subgroup medulloblastoma. Gorlin's syndrome precludes the use of radiotherapy (a standard component of treatment) due to the development of multiple basal cell carcinomas. Also, current SHH inhibitors are ineffective against SUFU-mutated medulloblastoma, as they inhibit upstream genes. In this study, we aimed to detect differences in the expression of genes and microRNAs between SUFU- and PTCH1-mutated SHH medulloblastomas which may hint at new treatment directions. PATIENTS AND METHODS: We sequenced RNA and microRNA from tumors of two patients with germline Gorlin's syndrome - one having PTCH1 mutation and one with SUFU mutation - followed by bioinformatics analysis to detect changes in genes and miRNAs expression in these two tumors. Expression changes were validated using qRT-PCR. Ingenuity pathway analysis was performed in search for targetable pathways. RESULTS: Compared to the PTCH1 tumor, the SUFU tumor demonstrated lower expression of miR-301a-3p and miR-181c-5p, matrix metallopeptidase 11 (MMP11) and OTX2, higher expression of miR-7-5p and corresponding lower expression of its targeted gene, connexin 30 (GJB6). We propose mechanisms to explain the phenotypic differences between the two types of tumors, and understand why PTCH1 and SUFU tumors tend to relapse locally (rather than metastatically as in other medulloblastoma subgroups). CONCLUSION: Our results help towards finding new treatable molecular targets for these types of medulloblastomas.
Assuntos
Neoplasias Cerebelares/genética , Mutação em Linhagem Germinativa , Meduloblastoma/genética , MicroRNAs/biossíntese , Receptor Patched-1/genética , RNA Neoplásico/biossíntese , Proteínas Repressoras/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/patologia , MicroRNAs/genética , Receptor Patched-1/metabolismo , RNA Neoplásico/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND/AIM: Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. MATERIALS AND METHODS: Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. RESULTS: A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. CONCLUSION: MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.
Assuntos
Neoplasias da Mama/genética , Proteínas Mad2/genética , Mitose/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Prometáfase/genéticaRESUMO
Unlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle [1] and is incorporated into chromatin independent of replication [2]. Recently, H3.3 has been implicated in the cellular response to ultraviolet (UV) light [3]. Here, we show that chicken DT40 cells completely lacking H3.3 are hypersensitive to UV light, a defect that epistasis analysis suggests may result from less-effective nucleotide excision repair. Unexpectedly, H3.3-deficient cells also exhibit a substantial defect in maintaining replication fork progression on UV-damaged DNA, which is independent of nucleotide excision repair, demonstrating a clear requirement for H3.3 during S phase. Both the UV hypersensitivity and replication fork slowing are reversed by expression of H3.3 and require the specific residues in the α2 helix that are responsible for H3.3 binding its dedicated chaperones. However, expression of an H3.3 mutant in which serine 31 is replaced with alanine, the equivalent residue in H3.2, restores normal fork progression but not UV resistance, suggesting that H3.3[S31A] may be incorporated at UV-damaged forks but is unable to help cells tolerate UV lesions. Similar behavior was observed with expression of H3.3 carrying mutations at K27 and G34, which have been reported in pediatric brain cancers. We speculate that incorporation of H3.3 during replication may mark sites of lesion bypass and, possibly through an as-yet-unidentified function of the N-terminal tail, facilitate subsequent processing of the damage.
Assuntos
Proteínas Aviárias/metabolismo , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Histonas/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular Tumoral , Galinhas , Cromatina/metabolismo , Dano ao DNARESUMO
The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/C(CDC20) ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/C(CDH1) has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/C(CDC20), releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/C(CDH1) substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/C(CDC20) and APC/C(CDH1) during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Anáfase , Caderinas/metabolismo , Proteínas Mad2/metabolismo , Mitose , Antígenos CD , Caderinas/genética , Proteínas Cdc20/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Mad2/genética , Fosforilação , Prometáfase , Proteólise , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , UbiquitinaçãoRESUMO
The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.