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Francis Beaudry - One of the best experts on this subject based on the ideXlab platform.
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liquid chromatography electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in TAC1 mouse spinal cords reveals significant lower concentration of opioid peptides
Neuropeptides, 2015Co-Authors: Mouna Saidi, Francis BeaudryAbstract:Abstract Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in TAC1 −/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the TAC1 −/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in TAC1 −/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated ( p −/− spinal cords for Met-Enk and CGRP. The analysis of TAC1 −/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.
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Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in TAC1(-/-) mouse spinal cords reveals significant lower concentration of opioid peptides.
Neuropeptides, 2015Co-Authors: Mouna Saidi, Francis BeaudryAbstract:Abstract Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in TAC1 −/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the TAC1 −/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in TAC1 −/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated ( p −/− spinal cords for Met-Enk and CGRP. The analysis of TAC1 −/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.
Lawrence C. Myers - One of the best experts on this subject based on the ideXlab platform.
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Candida albicans Zn Cluster Transcription Factors TAC1 and Znc1 Are Activated by Farnesol To Upregulate a Transcriptional Program Including the Multidrug Efflux Pump CDR1.
Antimicrobial agents and chemotherapy, 2018Co-Authors: Zhongle Liu, John M. Rossi, Lawrence C. MyersAbstract:Farnesol, a quorum-sensing molecule, inhibits Candida albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol's mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these processes are linked through the Zn cluster transcription factors TAC1 and Znc1 and their induction of the multidrug efflux pump Cdr1. Specifically, we have demonstrated that TAC1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1, ZNC1, or both. A related molecule, 1-dodecanol, invokes a similar TAC1-ZNC1 response, while several other proposed C. albicans quorum-sensing molecules do not. TAC1 and Znc1 both bind to and upregulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to TAC1 and Znc1 having overlapping, but nonidentical, regulons. Induction of genes by farnesol via TAC1 and Znc1 was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by TAC1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1, facilitates growth in the presence of high farnesol concentrations in C. albicans and in certain strains of its close relative, C. dubliniensis.
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C. albicans Zn Cluster Transcription Factors TAC1 and Znc1 are Activated by Farnesol to Up Regulate a Transcriptional Program Including the Multi-Drug Efflux Pump CDR1
2018Co-Authors: Zhongle Liu, John M. Rossi, Lawrence C. MyersAbstract:Farnesol, a quorum-sensing molecule, inhibits C. albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol9s mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these themes are linked through the Zn cluster transcription factors TAC1 and Znc1, and their induction of the multi-drug efflux pump Cdr1. Specifically, we have demonstrated that TAC1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1 , ZNC1 , or both. A related molecule, 1-dodecanol, invokes a similar TAC1/ZNC1 response, while several other proposed C. albicans quorum sensing molecules do not. TAC1 and ZNC1 both bind to and up-regulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to TAC1 and Znc1 having overlapping, but non-identical, regulons. TAC1 and ZNC1 dependent farnesol induction of their target genes was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by TAC1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1 , facilitates growth in the presence of high farnesol concentrations in C. albicans , and certain strains of its close relative C. dubliniensis .
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Mediator Tail Module Is Required for TAC1-Activated CDR1 Expression and Azole Resistance in Candida albicans.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Zhongle Liu, Lawrence C. MyersAbstract:ABSTRACT The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor TAC1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate TAC1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of TAC1. GOF mutations within the TAC1 TAD, outside the context of full-length TAC1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the TAC1 TAD by the TAC1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length TAC1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of TAC1 target genes, such as CDR1 , are dependent on the TAC1 TAD and subunits of the Mediator tail module. The dependence of different TAC1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of TAC1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for TAC1 hyperactivation. The role of Mediator in events downstream of TAC1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention.
Pranela Rameshwar - One of the best experts on this subject based on the ideXlab platform.
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The RNA-binding protein Musashi 1 stabilizes the oncotachykinin 1 mRNA in breast cancer cells to promote cell growth
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015Co-Authors: George R. Nahas, Raghav G. Murthy, Shyam A. Patel, Steven J. Greco, Teja Ganta, Pranela RameshwarAbstract:Substance P and its truncated receptor exert oncogenic effects. The high production of substance P in breast cancer cells (BCCs) is caused by the enhancement of tachykinin (TAC)1 translation by cytosolic factor. In vitro translational studies and mRNA stabilization analyses indicate that BCCs contain the factor needed to increase TAC1 translation and to stabilize the mRNA. Prediction of protein folding, RNA-shift analysis, and proteomic analysis identified a 40 kDa molecule that interacts with the noncoding exon 7. Western blot analysis and RNA supershift identified Musashi 1 (Msi1) as the binding protein. Ectopic expression of TAC1 in nontumorigenic breast cells (BCs) indicates that TAC1 regulates its stability by increasing Msi1. Using a reporter gene system, we showed that Msi1 competes with microRNA (miR)130a and -206 for the 3' UTR of exon 7/TAC1. In the absence of Msi1 and miR130a and -206, reporter gene activity decreased, indicating that Msi1 expression limits TAC1 expression. Tumor growth was significantly decreased when nude BALB/c mice were injected with Msi1-knockdown BCCs. In summary, the RNA-binding protein Msi1 competes with miR130a and -206 for interaction with TAC1 mRNA, to stabilize and increase its translation. Consequently, these interactions increase tumor growth.
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Abstract 1396: Musashi 1 stabilizes TAC1 transcript in breast cancer cells to increase the production of onco-substance P
Molecular and Cellular Biology, 2014Co-Authors: George R. Nahas, Raghav G. Murthy, Shyam A. Patel, Steven J. Greco, Pranela RameshwarAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite improved education and aggressive treatments, the mortality of breast cancer (BC) remains high. The TAC1 gene exerts oncogenic effects partly due to its major peptide, substance P, inducing the production of cytokines, which facilitate expression of the tumor-promoting truncated neurokinin-1 receptor. In turn, TAC1 facilitates metastasis to bone marrow and brain. TAC1 expression in BC cells (BCCs) occurs partly through increased translation of its mRNA. Cytosolic extracts from TAC1-expressing cells (BCCs and ectopic expression in non-tumorigenic breast cells) were analyzed for the candidate RNA-binding protein. In vitro translational, RNA stabilization, RNA shift and proteomic analyses identified a 40 kDa binding protein, which interacted with Exon 7 of the 3′ UTR of TAC1. Western blots and RNA supershift assay identified musashi 1 (Msi1) as the TAC1 mRNA binding protein. The data also indicated that TAC1 expression was sufficient for an increase in Msi1. A reporter gene system utilizing the TAC1 3′ UTR indicated that Msi1 competed with the endogenous translational suppressors, miR-130a and -206 for the same interacting site. Use of anti-miRs, Msi1 shRNA and the reporter gene system confirmed Msi1 as the stabilizer of TAC1 mRNA to increase the production of substance P. In vivo studies with nude mice showed reduced tumor growth with Msi1 knockdown BCCs compared to control. Msi1 knockdown retracted the tumor, indicating a role in tumor initiation. These findings correlated with a loss of two stem cell genes, REST and Oct4. In summary, this study identified Msi1 as a central RNA binding protein, which enhanced expression of the oncogenic TAC1 in BCCs. The data also suggested that Msi1, through TAC1 might be important in tumor initiation. These findings have clinical implications as a new drug target to reverse the oncogenic effects of TAC1. Furthermore, the feasibility of targeting TAC1 is evident since its receptor antagonist such as aprepitant are already approved by the FDA. Citation Format: George R. Nahas, Raghav G. Murthy, Shyam A. Patel, Steven J. Greco, Pranela Rameshwar. Musashi 1 stabilizes TAC1 transcript in breast cancer cells to increase the production of onco-substance P. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2014-1396
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Abstract 3198: The RNA-binding protein Musashi-1 stabilizes TAC1 mRNA in breast cancer cells.
Molecular and Cellular Biology, 2013Co-Authors: George R. Nahas, Raghav G. Murthy, Steven J. Greco, Pranela RameshwarAbstract:Breast cancer (BC) remains a major public health challenge in the United States. Prognosis for metastatic cancer is poor with nearly all deaths from BC due to metastatic disease. Upon discovery in bone, the five year survival rate of BC patients is approximately eight percent. The tachykinin gene, TAC1, has been implicated in facilitating metastasis of BC to the bone marrow. The predominant product of TAC1 is the neuropeptide, substance P (SP). We have previously demonstrated that in non-malignant cells, TAC1 translation is suppressed by miRNAs. Stimulation with specific cytokines decreased these miRNA to allow synthesis of SP. BC cells (BCCs) lack endogenous expression of the TAC1-specific miRNA, which is consistent with the increased levels of SP in these cells. Herein the present study investigated the translational control of TAC1 mRNA in BCCs. Cytoplasmic extracts from BCCs showed an increase in TAC1 translation and stabilization of its mRNA. These findings led us to bioinformatic analyses of the secondary structure of TAC1 mRNA, which predicted candidate interacting sites in Exons 3 and 7. These predictions were further refined by results from an RNA shift assay that indicated an RNA-binding protein specifically targeting Exon 7. Through western blots and proteomic analysis we identified the RNA-binding protein, Musashi-1 (Msi1), as a putative candidate. Identity was confirmed by RNA supershift assay utilizing an antibody specific for Msi1. Gain-of-function and reporter gene assays utilizing the Msi1 expression vector co-transfected with a luciferase reporter gene linked to the 3′UTR of TAC1, including Exon 7, confirmed a stabilizing role for Msi1 in TAC1 mRNA regulation, concomitant with an increase in substance P. In summary, we identified Msi1 as a novel binding protein for the oncogenic TAC1 mRNA. These findings have implications for the identification of novel targets to treat BC. Citation Format: George R. Nahas, Raghav G. Murthy, Steven J. Greco, Pranela Rameshwar. The RNA-binding protein Musashi-1 stabilizes TAC1 mRNA in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2013-3198
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RE-1–silencing transcription factor shows tumor-suppressor functions and negatively regulates the oncogenic TAC1 in breast cancer cells
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Bobby Y. Reddy, Steven J. Greco, Prem S. Patel, Katarzyna A. Trzaska, Pranela RameshwarAbstract:Breast cancer remains the most prevalent cancer among women in the United States. Substance P, a peptide derived from the TAC1 gene, mediates oncogenic properties in breast and other cancers. TAC1 expression facilitates the entry of breast cancer cells into bone marrow. The transcriptional repressor element 1-silencing transcription factor (REST) has been implicated in both oncogenic and tumor-suppressor functions. REST binds to the 5' untranslated region of the TAC1 promoter and suppresses its expression. This study investigated a role for REST in TAC1 induction in breast cancer. Western blots and real-time PCR indicated that REST expression in breast cancer cells was inversely proportional to the cells' aggressiveness, for both cell lines and primary breast cancer cells. REST knockdown in low-metastatic T47D cells and nontumorigenic MCF12A cells resulted in increases in TAC1 induction, proliferation, and migration. These parameters were negatively affected by ectopic expression of REST in highly aggressive MDA-MB-231 cells. Together, these findings show a central role for REST in the oncogenic function of TAC1 and suggest a tumor-suppressor role for REST in breast cancer.
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Stromal-derived factor-1α induces a non-canonical pathway to activate the endocrine-linked TAC1 gene in non-tumorigenic breast cells
Journal of molecular endocrinology, 2008Co-Authors: Kelly E. Corcoran, Ashwani Malhotra, Carlos A. Molina, Pranela RameshwarAbstract:The chemokine Stromal-derived factor-1alpha (SDF-1alpha) interacts with seven transmembrane (TM) G-protein-coupled receptor (GPR), CXCR4. SDF-1alpha is linked to inflammation, chemoattraction, cancer metastasis, and hematopoiesis. Tachykinin (TAC1) peptides bind seven transmembrane (TM), GPR and are involved in tumor promotion. SDF-1alpha regulates TAC1 expression in non-tumorigenic breast cells through a bimodal pattern with repression at high levels through nuclear factor-kappa B (NFkappaB) activation. This study focuses on the mechanism of activation at low SDF-1alpha in MCF12A non-tumorigenic breast cells. Reporter gene assays with the 5' flanking region of TAC1 (exon 1 omitted) and co-transfection with the repressor of cAMP response element (CREB) (ICER), and transfection with the CRE sites mutated, verified critical roles for CRE sites in SDF-1alpha-mediated TAC1 activation. Western blots and functional assays with specific inhibitors indicated that SDF-1alpha phosphorylated CREB (P-CREB) via Galpha(i)2-PI3K-protein kinase C (PKC)zeta-p38-extracellular signal-regulated kinase (ERK) and no evidence of cAMP-PKA pathway. This observation is different from previous studies that reported CREB-phosphorylated PKA pathway in the activation of TAC1 in bone marrow stromal cells. This suggests cell specificity in TAC1 expression. In conclusion, this study reports on a non-canonical pathway in TAC1 activation by SDF-1alpha. This finding is significant, since TAC1 is relevant to breast cancer metastasis, to bone marrow where stromal cells have a significant facilitating function.
Mouna Saidi - One of the best experts on this subject based on the ideXlab platform.
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liquid chromatography electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in TAC1 mouse spinal cords reveals significant lower concentration of opioid peptides
Neuropeptides, 2015Co-Authors: Mouna Saidi, Francis BeaudryAbstract:Abstract Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in TAC1 −/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the TAC1 −/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in TAC1 −/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated ( p −/− spinal cords for Met-Enk and CGRP. The analysis of TAC1 −/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.
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Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in TAC1(-/-) mouse spinal cords reveals significant lower concentration of opioid peptides.
Neuropeptides, 2015Co-Authors: Mouna Saidi, Francis BeaudryAbstract:Abstract Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in TAC1 −/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the TAC1 −/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in TAC1 −/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated ( p −/− spinal cords for Met-Enk and CGRP. The analysis of TAC1 −/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.
Zhongle Liu - One of the best experts on this subject based on the ideXlab platform.
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Candida albicans Zn Cluster Transcription Factors TAC1 and Znc1 Are Activated by Farnesol To Upregulate a Transcriptional Program Including the Multidrug Efflux Pump CDR1.
Antimicrobial agents and chemotherapy, 2018Co-Authors: Zhongle Liu, John M. Rossi, Lawrence C. MyersAbstract:Farnesol, a quorum-sensing molecule, inhibits Candida albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol's mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these processes are linked through the Zn cluster transcription factors TAC1 and Znc1 and their induction of the multidrug efflux pump Cdr1. Specifically, we have demonstrated that TAC1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1, ZNC1, or both. A related molecule, 1-dodecanol, invokes a similar TAC1-ZNC1 response, while several other proposed C. albicans quorum-sensing molecules do not. TAC1 and Znc1 both bind to and upregulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to TAC1 and Znc1 having overlapping, but nonidentical, regulons. Induction of genes by farnesol via TAC1 and Znc1 was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by TAC1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1, facilitates growth in the presence of high farnesol concentrations in C. albicans and in certain strains of its close relative, C. dubliniensis.
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C. albicans Zn Cluster Transcription Factors TAC1 and Znc1 are Activated by Farnesol to Up Regulate a Transcriptional Program Including the Multi-Drug Efflux Pump CDR1
2018Co-Authors: Zhongle Liu, John M. Rossi, Lawrence C. MyersAbstract:Farnesol, a quorum-sensing molecule, inhibits C. albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol9s mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these themes are linked through the Zn cluster transcription factors TAC1 and Znc1, and their induction of the multi-drug efflux pump Cdr1. Specifically, we have demonstrated that TAC1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1 , ZNC1 , or both. A related molecule, 1-dodecanol, invokes a similar TAC1/ZNC1 response, while several other proposed C. albicans quorum sensing molecules do not. TAC1 and ZNC1 both bind to and up-regulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to TAC1 and Znc1 having overlapping, but non-identical, regulons. TAC1 and ZNC1 dependent farnesol induction of their target genes was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by TAC1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1 , facilitates growth in the presence of high farnesol concentrations in C. albicans , and certain strains of its close relative C. dubliniensis .
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Mediator Tail Module Is Required for TAC1-Activated CDR1 Expression and Azole Resistance in Candida albicans.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Zhongle Liu, Lawrence C. MyersAbstract:ABSTRACT The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor TAC1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate TAC1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of TAC1. GOF mutations within the TAC1 TAD, outside the context of full-length TAC1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the TAC1 TAD by the TAC1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length TAC1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of TAC1 target genes, such as CDR1 , are dependent on the TAC1 TAD and subunits of the Mediator tail module. The dependence of different TAC1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of TAC1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for TAC1 hyperactivation. The role of Mediator in events downstream of TAC1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention.
