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1.
PLoS Genet ; 20(6): e1011302, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38829899

RESUMO

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.


Assuntos
Proteínas de Ciclo Celular , Cryptococcus neoformans , Proteínas Mad2 , Fuso Acromático , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Mad2/metabolismo , Proteínas Mad2/genética , Fuso Acromático/metabolismo , Fuso Acromático/genética , Transdução de Sinais , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/genética , Cinetocoros/metabolismo , Segregação de Cromossomos/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
2.
EMBO Rep ; 25(6): 2743-2772, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38806674

RESUMO

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.


Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Proteína 11 Semelhante a Bcl-2 , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Mad2 , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Camundongos , Proteínas Mad2/metabolismo , Proteínas Mad2/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Atrofia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Mitose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Medula Óssea/patologia , Medula Óssea/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Supressoras de Tumor
3.
Cell Signal ; 119: 111172, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38604342

RESUMO

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.


Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Sinvastatina , Sinvastatina/farmacologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Células HeLa , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mitose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo
4.
Chromosoma ; 133(2): 149-168, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38456964

RESUMO

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.


Assuntos
Proteínas Cromossômicas não Histona , Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Espermatócitos , Animais , Masculino , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Ratos , Espermatócitos/metabolismo , Espermatócitos/citologia , Pontos de Checagem da Fase M do Ciclo Celular/genética , Espermatogênese , Testículo/metabolismo , Testículo/citologia , Cinesinas/metabolismo , Cinesinas/genética , Fuso Acromático/metabolismo
5.
Development ; 151(6)2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38546043

RESUMO

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.


Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Animais , Feminino , Camundongos , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Oócitos/metabolismo , Ubiquitinas/metabolismo
6.
PLoS One ; 19(3): e0301084, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38530809

RESUMO

There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Antifúngicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Estudos Prospectivos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo
7.
Proc Natl Acad Sci U S A ; 121(12): e2322677121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38466841

RESUMO

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.


Assuntos
Arabidopsis , Cinetocoros , Animais , Cinetocoros/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem do Ciclo Celular , Fuso Acromático/metabolismo
8.
Mol Cancer Res ; 22(5): 423-439, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38324016

RESUMO

NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.


Assuntos
Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Mitose , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Fuso Acromático/metabolismo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética
9.
Prostate ; 84(6): 605-619, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38375594

RESUMO

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.


Assuntos
Cisplatino , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Cisplatino/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Tubulina (Proteína)/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Linhagem Celular Tumoral , Apoptose , Proliferação de Células , Microtúbulos/metabolismo , Microtúbulos/patologia , Desacetilase 6 de Histona/metabolismo
10.
Commun Biol ; 7(1): 164, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38337031

RESUMO

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.


Assuntos
Proteínas Cdc20 , Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/química , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Transdução de Sinais , Humanos
11.
Clin Lab ; 70(1)2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38213208

RESUMO

BACKGROUND: BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is a member of the spindle assembly checkpoint family and is related to cancer disease progression, invasion, metastasis, and functional promotion of angiogenesis. Several studies have noted that the BUB1B gene is frequently upregulated in various types of cancers. However, the expression patterns of BUB1B across different cancer types and its diagnostic and prognostic potential have not been investigated from a pan-cancer perspective. METHODS: The Cancer Genome Atlas (TCGA) data were used to explore the diagnostic and prognostic immunological potential of BUB1B in 33 cancer types. RESULTS: BUB1B was almost universally upregulated across all cancers, with increased protein expression in at least six cancer types and an enhanced phosphorylation level of S670 in two cancer types. Furthermore, BUB1B expression was negatively associated with clinical progression and prognosis in most cancers. BUB1B expression was positively associated with tumor mutational burden and microsatellite instability in 17 and 7 cancer types, respectively, and there was a correlation between BUB1B expression and DNA methylation at multiple probes in 30 cancer types. Additionally, a positive relationship existed between BUB1B expression and the infiltration levels of Th2, Tcm, and T helper cells, whereas BUB1B showed a negative correlation with the infiltration levels of other immune cells in multiple cancers. Moreover, functions associated with cell cycle progression and ubiquitin-mediated proteolysis were involved in the functional mechanism of BUB1B. CONCLUSIONS: Our pan-cancer study offers a comprehensive understanding of the role of BUB1B in tumorigenesis and tumor immunity across different types of cancer.


Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Neoplasias , Humanos , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Neoplasias/diagnóstico , Neoplasias/genética , Serina , Proteínas de Ciclo Celular
12.
Proc Natl Acad Sci U S A ; 121(2): e2316583121, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38170753

RESUMO

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mitose , Cinetocoros/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Segregação de Cromossomos
13.
Haematologica ; 109(2): 578-590, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37496433

RESUMO

Despite substantial recent advances in treatment, multiple myeloma (MM) remains an incurable disease, with a shortage of treatment options for patients with high-risk disease, warranting the need for novel therapeutic targets and treatment approaches. Threonine and tyrosine kinase (TTK), also known as monopolar spindle 1 (MPS1), is a kinase essential for the mitotic spindle checkpoint whose expression correlates to unfavorable prognosis in several cancers. Here, we report the importance of TTK in MM, and the effects of the TTK inhibitor OSU-13. Elevated TTK expression correlated with amplification/ gain of 1q21 and decreased overall and event-free survival in MM. Treatment with OSU-13 inhibited TTK activity efficiently and selectively at a similar concentration range to other TTK inhibitor clinical candidates. OSU-13 reduced proliferation and viability of primary human MM cells and cell lines, especially those with high 1q21 copy numbers, and triggered apoptosis through caspase 3 and 7 activation. In addition, OSU-13 induced DNA damage and severe defects in chromosome alignment and segregation, generating aneuploidy. In vivo, OSU-13 decreased tumor growth in mice with NCI-H929 xenografts. Collectively, our findings reveal that inhibiting TTK with OSU-13 is a potential therapeutic strategy for MM, particularly for a subset of high-risk patients with poor outcome.


Assuntos
Proteínas de Ciclo Celular , Mieloma Múltiplo , Humanos , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases , Linhagem Celular Tumoral
14.
Chromosoma ; 133(1): 77-92, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37256347

RESUMO

Chromosome gains or losses often lead to copy number variations (CNV) and loss of heterozygosity (LOH). Both quantities are low in hematologic "liquid" cancers versus solid tumors in data of The Cancer Genome Atlas (TCGA) that also shows the fraction of a genome affected by LOH is ~ one-half of that with CNV. Suspension cultures of p53-null THP-1 leukemia-derived cells conform to these trends, despite novel evidence here of genetic heterogeneity and transiently elevated CNV after perturbation. Single-cell DNAseq indeed reveals at least 8 distinct THP-1 aneuploid clones with further intra-clonal variation, suggesting ongoing genetic evolution. Importantly, acute inhibition of the mitotic spindle assembly checkpoint (SAC) produces CNV levels that are typical of high-CNV solid tumors, with subsequent cell death and down-selection to novel CNV. Pan-cancer analyses show p53 inactivation associates with aneuploidy, but leukemias exhibit a weaker trend even though p53 inactivation correlates with poor survival. Overexpression of p53 in THP-1 does not rescue established aneuploidy or LOH but slightly increases cell death under oxidative or confinement stress, and triggers p21, a key p53 target, but without affecting net growth. Our results suggest that factors other than p53 exert stronger pressures against aneuploidy in liquid cancers, and identifying such CNV suppressors could be useful across liquid and solid tumor types.


Assuntos
Leucemia , Neoplasias , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Variações do Número de Cópias de DNA , Heterogeneidade Genética , Aneuploidia , Neoplasias/genética , Neoplasias/metabolismo , Leucemia/genética , Leucemia/metabolismo , Fuso Acromático/metabolismo
15.
J Clin Invest ; 134(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37934606

RESUMO

Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in Caenorhabditis elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.


Assuntos
Neoplasias da Próstata , Mutações Sintéticas Letais , Animais , Humanos , Masculino , Proteína BRCA2 , Caenorhabditis elegans/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mamíferos/metabolismo , Mutação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Ubiquitina Tiolesterase/genética , Proteína Fosfatase 2/metabolismo
16.
Nat Cancer ; 5(1): 66-84, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38151625

RESUMO

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.


Assuntos
Cinesinas , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Cinesinas/genética , Cinesinas/metabolismo , Mitose/genética , Linhagem Celular , Pontos de Checagem da Fase M do Ciclo Celular
17.
FEBS Lett ; 598(2): 252-265, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38112379

RESUMO

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.


Assuntos
Carcinoma Adenoide Cístico , Humanos , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Pontos de Checagem da Fase M do Ciclo Celular , Fatores de Transcrição/genética , Glândulas Salivares , Proteínas Serina-Treonina Quinases/genética
18.
EMBO Rep ; 24(11): e57227, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37795949

RESUMO

Chromosome segregation errors in mammalian oocyte meiosis lead to developmentally compromised aneuploid embryos and become more common with advancing maternal age. Known contributors include age-related chromosome cohesion loss and spindle assembly checkpoint (SAC) fallibility in meiosis-I. But how effective the SAC is in meiosis-II and how this might contribute to age-related aneuploidy is unknown. Here, we developed genetic and pharmacological approaches to directly address the function of the SAC in meiosis-II. We show that the SAC is insensitive in meiosis-II oocytes and that as a result misaligned chromosomes are randomly segregated. Whilst SAC ineffectiveness in meiosis-II is not age-related, it becomes most prejudicial in oocytes from older females because chromosomes that prematurely separate by age-related cohesion loss become misaligned in meiosis-II. We show that in the absence of a robust SAC in meiosis-II these age-related misaligned chromatids are missegregated and lead to aneuploidy. Our data demonstrate that the SAC fails to prevent cell division in the presence of misaligned chromosomes in oocyte meiosis-II, which explains how age-related cohesion loss can give rise to aneuploid embryos.


Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Feminino , Animais , Fuso Acromático/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Meiose/genética , Oócitos , Cromátides , Aneuploidia , Segregação de Cromossomos , Mamíferos/genética
19.
EMBO J ; 42(20): e112630, 2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37712330

RESUMO

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.


Assuntos
Cinetocoros , Pontos de Checagem da Fase M do Ciclo Celular , Humanos , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Fosforilação , Microtúbulos/metabolismo , Mitose , Células HeLa
20.
Curr Biol ; 33(20): 4505-4515.e4, 2023 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-37738972

RESUMO

During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation.1,2,3,4 The signaling activity of these kinetochores and the likelihood of chromosome missegregation depend on the amount of SAC signaling proteins each kinetochore recruits.5,6,7,8 Therefore, factors that control SAC protein recruitment must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins due to the concerted action of many kinases and phosphatases is a significant determinant of the SAC protein recruitment to signaling kinetochores.9 Whether the abundance of SAC proteins also influences the recruitment and signaling activity of human kinetochores has not been studied.8,10 Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits its own recruitment and that of BubR1 and restricts the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher recruitment of SAC proteins, producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins, including Bub1. The inverse correlation may allow the dividing cell to prevent a large number of signaling kinetochores in early prophase from generating an overly large signal while enabling the last unaligned kinetochore in late prometaphase to signal at the maximum strength.


Assuntos
Proteínas de Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transdução de Sinais , Cinetocoros/metabolismo , Mitose , Fuso Acromático/metabolismo
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