RESUMO
Regulation of actomyosin ring contraction is important for the coordination of cytokinesis with mitosis. Hof1, a member of the Pombe Cdc15 homology (PCH) family of proteins, is required for efficient cytokinesis in budding yeast. Phosphorylation of Hof1 depends on the mitotic exit network (MEN), and its degradation at the end of mitosis depends on its PEST motif and interaction with the E3 ligase Grr1. To test the hypothesis that targeted destruction of Hof1 temporally couples mitotic exit with contraction of the actomyosin ring, we mutated the Hof1 PEST motif to prevent phosphorylation and subsequent degradation. These mutations increased the amount of Hof1 at the bud neck during cytokinesis, resulted in smaller bud neck diameter, and slowed the rate of myosin contraction. However, Hof1 PEST motif phosphorylation site mutants did not have cytokinesis defects, indicating that regulation of Hof1 levels does not control the onset of actomyosin ring contraction as predicted.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Citocinese , Proteínas F-Box/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Miosinas/fisiologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chemotherapy-induced neuropathic pain (CINP) in both sexes compromises many current chemotherapeutics and lacks an FDA-approved therapy. We recently identified the sphingosine-1-phosphate receptor subtype 1 (S1PR1) and A3 adenosine receptor subtype (A3AR) as novel targets for therapeutic intervention. Our work in male rodents using paclitaxel, oxaliplatin, and bortezomib showed robust inhibition of CINP with either S1PR1 antagonists or A3AR agonists. The S1PR1 functional antagonist FTY720 (Gilenya) is FDA-approved for treating multiple sclerosis, and selective A3AR agonists are in advanced clinical trials for cancer and inflammatory disorders, underscoring the need for their expedited trials in patients with CINP as chemotherapy adjuncts. Our findings reveal that S1PR1 antagonists and A3AR agonists mitigate paclitaxel and oxaliplatin CINP in female and male rodents, but failed to block or reverse bortezomib-induced neuropathic pain (BINP) in females. Although numerous mechanisms likely underlie these differences, we focused on receptor levels. We found that BINP in male rats, but not in female rats, was associated with increased expression of A3AR in the spinal cord dorsal horn, whereas S1PR1 levels were similar in both sexes. Thus, alternative mechanisms beyond receptor expression may account for sex differences in response to S1PR1 antagonists. Morphine and duloxetine, both clinical analgesics, reversed BINP in female mice, demonstrating that the lack of response is specific to S1PR1 and A3AR agents. Our findings suggest that A3AR- and S1PR1-based therapies are not viable approaches in preventing and treating BINP in females and should inform future clinical trials of these drugs as adjuncts to chemotherapy.
Assuntos
Antineoplásicos/efeitos adversos , Bortezomib/efeitos adversos , Neuralgia/tratamento farmacológico , Receptor A3 de Adenosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Antagonistas do Receptor A3 de Adenosina/administração & dosagem , Antagonistas do Receptor A3 de Adenosina/uso terapêutico , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Animais , Cloridrato de Duloxetina/administração & dosagem , Cloridrato de Duloxetina/uso terapêutico , Feminino , Cloridrato de Fingolimode/administração & dosagem , Cloridrato de Fingolimode/uso terapêutico , Masculino , Morfina/administração & dosagem , Morfina/uso terapêutico , Neuralgia/induzido quimicamente , Oxaliplatina/efeitos adversos , Paclitaxel/efeitos adversos , Ratos , Fatores Sexuais , Moduladores do Receptor de Esfingosina 1 Fosfato/administração & dosagem , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico , Corno Dorsal da Medula Espinal/efeitos dos fármacosRESUMO
BACKGROUND: The mitotic exit network (MEN) is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP) to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction. RESULTS: To test the hypothesis that the Bub2 pathway prevents premature actin ring assembly, we compared the timing of actin ring formation in wild type, bub2Delta, mad2Delta, and bub2Deltamad2Delta cells both with and without microtubules. There was no difference in the timing of actin ring formation between wild type and mutant cells in a synchronized cell cycle. In the presence of nocodazole, both bub2Delta and mad2Delta cells formed rings after a delay of the same duration. Double mutant bub2Deltamad2Delta and bfa1Deltamad2Delta cells formed rings at the same time with and without nocodazole. To determine if Bub2 has an effect on actomyosin ring contraction through its regulation of Tem1, we used live cell imaging of Myo1-GFP in a bub2Delta strain. We found a significant decrease in the total time of contraction and an increase in rate of contraction compared to wild type cells. We also examined myosin contraction using Myo1-GFP in cells overexpressing an epitope tagged Bub2. Surprisingly, overexpression of Bub2 also led to a significant increase in the rate of contraction, as well as morphological defects. The chained cell phenotype caused by Bub2 overexpression could be rescued by co-overexpression of Tem1, and was not rescued by deletion of BFA1. CONCLUSION: Our data indicate that the Bub2 checkpoint pathway does not have a specific role in delaying actin ring formation. The observed increase in the rate of myosin contraction in the bub2Delta strain provides evidence that the MEN regulates actomyosin ring contraction. Our data suggest that the overexpression of the Bub2 fusion protein acts as a dominant negative, leading to septation defects by a mechanism that is Tem1-dependent.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/biossíntese , Proteínas de Ciclo Celular/genética , Citocinese/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Genes Fúngicos/fisiologia , Miosinas/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The development of chemotherapy-induced painful peripheral neuropathy is a major dose-limiting side effect of many chemotherapeutics, including bortezomib, but the mechanisms remain poorly understood. We now report that bortezomib causes the dysregulation of de novo sphingolipid metabolism in the spinal cord dorsal horn to increase the levels of sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) ligands, S1P and dihydro-S1P. Accordingly, genetic and pharmacological disruption of S1PR1 with multiple S1PR1 antagonists, including FTY720, blocked and reversed neuropathic pain. Mice with astrocyte-specific alterations of S1pr1 did not develop neuropathic pain and lost their ability to respond to S1PR1 inhibition, strongly implicating astrocytes as a primary cellular substrate for S1PR1 activity. At the molecular level, S1PR1 engaged astrocyte-driven neuroinflammation and altered glutamatergic homeostasis, processes blocked by S1PR1 antagonism. Our findings establish S1PR1 as a target for therapeutic intervention and provide insight into cellular and molecular pathways. As FTY720 also shows promising anticancer potential and is FDA approved, rapid clinical translation of our findings is anticipated.