RESUMO
The cell wall integrity pathway (CWI) is a MAPK-mediated signaling route essential for yeast cell response to cell wall damage, regulating distinct aspects of fungal physiology. We have recently proven that the incorporation of a genetic circuit that operates as a signal amplifier into this pathway allows for the identification of novel elements involved in CWI signaling. Here, we show that the strong growth inhibition triggered by pathway hyperactivation in cells carrying the "Integrity Pathway Activation Circuit" (IPAC) also allows the easy identification of new stimuli. By using the IPAC, we have found various chemical agents that activate the CWI pathway, including the aminoglycoside neomycin. Cells lacking key components of this pathway are sensitive to this antibiotic, due to the disruption of signaling upon neomycin stimulation. Neomycin reduces both phosphatidylinositol-4,5-bisphosphate (PIP2) availability at the plasma membrane and myriocin-induced TORC2-dependent Ypk1 phosphorylation, suggesting a strong interference with plasma membrane homeostasis, specifically with PIP2. The neomycin-induced transcriptional profile involves not only genes related to stress and cell wall biogenesis, but also to amino acid metabolism, reflecting the action of this antibiotic on the yeast ribosome.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Aminoácidos/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Fosfatos de Inositol/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neomicina/farmacologia , Fosfatidilinositóis/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/MS/MS was used to study the sperm proteome under these two distinct conditions and bioinformatic enrichment analysis conducted. Gene Ontology (GO) and pathway enrichment analysis were performed revealing dramatic changes as consequence of cryopreservation. The terms oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport and electron transport chain were significantly enriched in fresh samples (Pâ¯=â¯5.50â¯×â¯10-12, 4.26â¯×â¯10-8 and 7.26â¯×â¯10-8, respectively), while were not significantly enriched in frozen thawed samples (Pâ¯=â¯1). The GO terms oxidation reduction process and oxidoreductase activity were enriched in fresh samples and the enrichment was reduced in frozen thawed samples (1.40â¯×â¯10-8, 1.69â¯×â¯10-6 versus 1.13â¯×â¯10-2 and 2-86â¯×â¯10-2 respectively). Reactome pathways (using human orthologs) significantly enriched in fresh sperm were TCA cycle and respiratory electron transport (Pâ¯=â¯1.867â¯×â¯10-8), Respiratory electron transport ATP synthesis by chemiosmosis coupling (Pâ¯=â¯2.124â¯×â¯10-5), Citric acid cycle (TCA cycle)(Pâ¯=â¯8.395â¯×â¯10-4) Pyruvate metabolism and TCA cycle (Pâ¯=â¯3.380â¯×â¯10-3), Respiratory electron transport (Pâ¯=â¯2.764â¯×â¯10-2) and Beta oxidation of laurolyl-CoA to decanoyl CoA-CoA (Pâ¯=â¯1.854â¯×â¯10-2) none of these pathways were enriched in thawed samples (Pâ¯=â¯1). We have provided the first detailed study on how the cryopreservation process impacts the stallion sperm proteome. Our findings identify the metabolic proteome and redoxome as the two key groups of proteins affected by the procedure. SIGNIFICANCE: In the present manuscript we investigated how the cryopreservation of stallion spermatozoa impacts the proteome of these cells. This procedure is routinely used in horse breeding and has a major impact in the industry, facilitating the trade of genetic material. This is still a suboptimal biotechnology, with numerous unresolved problems. The limited knowledge of the molecular insults occurring during cryopreservation is behind these problems. The application and development of proteomics to the spermatozoa, allow to obtain valuable information of the specific mechanisms affected by the procedure. In this paper, we report that cryopreservation impacts numerous proteins involved in metabolism regulation (mainly mitochondrial proteins involved in the TCA cycle, and oxidative phosphorylation) and also affects proteins with oxidoreductase activity. Moreover, specific proteins involved in the sperm-oocyte interaction are also affected by the procedure. The information gathered in this study, opens interesting questions and offer new lines of research for the improvement of the technology focusing the targets here identified, and the specific steps in the procedure (cooling, toxicity of antioxidants etc.) to be modified to reduce the damage.
Assuntos
Preservação do Sêmen , Animais , Criopreservação , Cavalos , Humanos , Masculino , Oxirredução , Proteômica , Motilidade dos Espermatozoides , Espermatozoides , Espectrometria de Massas em TandemRESUMO
The cell wall integrity (CWI) pathway mediates the response of Saccharomyces cerevisiae to cell wall alterations. Stress at the cell surface is detected by mechanosensors, which transduce the signal to a protein kinase cascade that involves Pkc1, Bck1, Mkk1/Mkk2, the mitogen-activated protein kinase (MAPK) Slt2 and the transcription factor Rlm1. We incorporated a positive feedback loop into this pathway by placing a hyperactive MKK1 allele under the control of the Rlm1-regulated MLP1 promoter. This circuit operates as a signal amplifier and leads to a highly increased Slt2 activation under stimulating conditions. Triggering the CWI pathway in cells engineered with this circuit, which we have named the Integrity Pathway Activation Circuit (IPAC), results in strong growth inhibition. Exploitation of this hypersensitive phenotype allowed the identification of novel proteins that contribute in signalling to Rlm1 in response to cell surface stressing agents such as Congo red, zymolyase and SDS. Among these proteins, the MAPK kinase kinase Ssk2 of the osmoregulatory high-osmolarity glycerol (HOG) pathway, but not its paralogue Ssk22, proved to be necessary for the SDS-induced IPAC-mediated growth inhibition. We found the existence of an Ssk1-independent Ssk2-Pbs2-Hog1-CWI pathway signalling axis that contributes to Slt2 activation in response to cell surface stress. We also demonstrated that the MAP kinase kinases Mkk1 and Pbs2 and the MAPKs Slt2 and Hog1 of the HOG and CWI pathways interact physically, forming a complex. Our results show how a simple synthetic circuit can be used as a powerful tool for a better understanding of signalling pathways.
Assuntos
Parede Celular/metabolismo , Retroalimentação Fisiológica , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/genética , Regulação Fúngica da Expressão Gênica , MAP Quinase Quinase Quinases/genética , Microscopia de Fluorescência , Osmorregulação/genética , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse MecânicoRESUMO
The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.
Assuntos
Quebras de DNA de Cadeia Dupla , Fosfoproteínas Fosfatases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Fosforilação , Fosfosserina/metabolismoRESUMO
Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion.
RESUMO
The yeast cell wall integrity MAPK Slt2 mediates the transcriptional response to cell wall alterations through phosphorylation of transcription factors Rlm1 and SBF. However, the variety of cellular functions regulated by Slt2 suggests the existence of a significant number of still unknown substrates for this kinase. To identify novel Slt2 targets, we generated and characterized an analog-sensitive mutant of Slt2 (Slt2-as) that can be specifically inhibited by bulky kinase inhibitor analogs. We demonstrated that Slt2-as is able to use adenosine 5'-[γ-thio]triphosphate analogs to thiophosphorylate its substrates in yeast cell extracts as well as when produced as recombinant proteins in Escherichia coli. Taking advantage of this chemical-genetic approach, we found that Slt2 phosphorylates the MAPK phosphatase Msg5 both in the N-terminal regulatory and C-terminal catalytic domains. Moreover, we identified the calcineurin regulator Rcn2, the 4E-BP (translation initiation factor eIF4E-binding protein) translation repressor protein Caf20, and the Golgi-associated adaptor Gga1 as novel targets for Slt2. The Slt2 phosphorylation sites on Rcn2 and Caf20 were determined. We also demonstrated that, in the absence of SLT2, the GGA1 paralog GGA2 is essential for cells to survive under cell wall stress and for proper protein sorting through the carboxypeptidase Y pathway. Therefore, Slt2-as provides a powerful tool that can expand our knowledge of the outputs of the cell wall integrity MAPK pathway.