The Neurospora crassa cps-1 gene encodes a polysaccharide synthase with homology to the Cryptococcus neoformans hyaluronic acid synthase Cps1p . Homologs of the cps-1 gene are found in the genomes of many fungi. Loss of CPS-1 results in a cell wall defect that affects all stages of the N. crassa life cycle, including vegetative growth, protoperithecia (female mating structure) development, and conidia (asexual spore) development. The cell wall of cps-1 deletion mutants is sensitive to cell wall perturbation reagents. Our results demonstrate that CPS-1 is required for the incorporation of cell wall proteins into the cell wall and plays a critical role in cell wall biogenesis. We found that the N. crassa cell wall is devoid of hyaluronic acid, and conclude that the polysaccharide produced by the CPS-1 is not hyaluronic acid.
Gene duplication promotes the diversification of protein functions in several ways. Ancestral functions can be partitioned between the paralogs, or a new function can arise in one paralog. These processes are generally viewed as unidirectional. However, paralogous proteins often retain related functions and can substitute for one another. Moreover, in the event of gene loss, the remaining paralog might regain ancestral functions that had been shed. To explore this possibility, we focused on the sirtuin deacetylase SIR2 and its homolog HST1 in the CTG clade of yeasts. HST1 has been consistently retained throughout the clade, whereas SIR2 is only present in a subset of species. These NAD+-dependent deacetylases generate condensed chromatin that represses transcription and stabilizes tandemly repeated sequences. By analyzing phylogenetic trees and gene order, we found that a single duplication of the SIR2/HST1 gene occurred, likely prior to the emergence of the CTG clade. This ancient duplication was followed by at least two independent losses of SIR2. Functional characterization of Sir2 and Hst1 in three species revealed that these proteins have not maintained consistent functions since the duplication. In particular, the rDNA locus is deacetylated by Sir2 in Candida albicans, by Hst1 in C. lusitaniae, and by neither paralog in C. parapsilosis. In addition, the subtelomeres in C. albicans are deacetylated by Sir2 rather than by Hst1, which is orthologous to the sirtuin associated with Saccharomyces cerevisiae subtelomeres. These differences in function support the model that sirtuin deacetylases can regain ancestral functions to compensate for gene loss.
Candida parapsilosis is one of the leading causes of hospital-acquired yeast infections and poses a significant risk to immunocompromised people. Two of its properties that contribute to infection are metabolic flexibility, to use a range of nutrients available in the host, and cellular dimorphism, to switch between round yeast cells and chains of elongated pseudohyphal cells.
Clear cell renal cell carcinoma (ccRCC) is the most common lethal subtype of kidney cancer. Large-scale metabolomics data have associated metabolic alterations with the pathogenesis and progression of renal cell carcinoma and have correlated mitochondrial activity with poor patient survival. Our group has recently reported that the lysosome purinergic receptor P2 × 4 (P2 × 4R) regulates neoangiogenesis. As ccRCC is characterized by increased angiogenesis we hypothesized that P2 × 4R may play a role in this disease. Interestingly, TCGA data suggest that P2 × 4R over-expression correlates with poor overall ccRCC patient survival. Our preliminary results also suggest that oxophosphorylation is the main source of tumor-derived ATP, which exerts a critical impact on tumor energy metabolism and mitochondrial activity in ccRCC. P2 × 4 receptor expression appears to contribute to the homeostasis of intracellular calcium and integrity of mitochondrial membrane potential in different ccRCC models. Seahorse experiments showed that P2 × 4R inhibition by 5BDBD, a potent and selective antagonist, caused a rapid dose-dependent reduction of mitochondrial activity. Moreover, prolonged mitochondrial failure induced by 5BDBD was associated with increased radical oxygen species, changes in mitochondrial permeability (i.e. opening of the transition pore complex, dissipation of membrane potential and calcium overload), and cell death via both necrosis and apoptosis. P2 × 4R inhibition by 5BDBD was associated with a significant anti-tumor effect both in vitro and in vivo by utilizing several ccRCC cell lines and patient-derived organoids. Interestingly, higher mitochondrial activity was associated with greater sensitivity to 5BDBD. Overall, our results suggest that the perturbed balance between lysosomal integrity and mitochondrial activity induced by P2 × 4R inhibition may be a therapeutic strategy for a subset of renal carcinoma patients Citation Format: Christopher Rupert, Roberto Pili, Filomena DeNigris. Therapeutic targeting of P2 × 4 receptor and mitochondrial membrane potential in renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4828.
Prostate cancer (PCa) is the most common cancer diagnosed in men in the Western countries. Epidemiological studies have suggested that environmental factors such as the Western diet, characterized by the consumption of high caloric food containing large amounts of animal protein and fats, play a key role in the pathogenesis of PCa. We propose that dietary intervention, by reducing caloric or protein intake, may be beneficial for the treatment of PCa. Our previous data suggests that dietary protein restriction reduces tumor growth in a LuCaP23.1 patient-derived xenograft (PDX) model of PCa. Thus, we tested the hypothesis whether caloric restriction induced by alternate-day fasting has a similar effect. Interestingly, caloric restriction had antitumor activity in the LuCaP23.1 model but only alternate-day fasting reduced the expression of androgen receptor (AR), a major driver of PCa growth. Therapies targeting AR activity, termed androgen deprivation therapies including the AR antagonist enzalutamide (ENZ), are the current standard of care for PCa. To address the therapeutic potential of combining alternate-day fasting with anti-androgens, we utilized the syngeneic mouse PCa xenograft models MyC-CaP and PTEN−/− RB−/−, and the human LuCaP23.1 PDX model. These AR-positive xenograft models harbor the oncogenic drivers most frequently found in PCa (i.e., MYC amplification and loss of the tumor suppressor PTEN). Here, we show that alternate-day fasting enhances the activity of ENZ in these PCa xenograft models. Interestingly, the combination of alternate-day fasting with ENZ treatment reduced AR expression and signaling, and decreased proliferation and tumor growth as compared to either single treatment alone. Consistent with these findings, the reduction of AR activity by the depletion of endogenous androgens following surgical castration was enhanced with alternate-day fasting. Conversely, dietary protein restriction had no effect in combination ENZ in vivo, suggesting that modulation of AR by alternate-day fasting enhances the effect of anti-androgens. Transcriptomic analysis of tumors from fasted mice confirmed decreased AR transcriptional activity and revealed that several nutrient sensitive pathways were reduced by this dietary regimen, including PI3K/AKT signaling, mTORC1 signaling, and glycolysis. Additionally, we observed that several known factors induced by fasting were upregulated in tumors from fasted mice, including SIRT1 which has been suggested to modulate AR transcriptional activity. In conclusion, we demonstrate that alternate-day fasting reduces AR signaling and enhances the activity of ENZ in several PCa xenograft models. Overall, this study suggests that caloric restriction may improve the efficacy of anti-androgen therapy in PCa patients. Citation Format: Ricardo Cordova, May Elbanna, Angela Klunk, Christopher Rupert, Li Shen, Yanqing Wang, David Goodrich, Ron Wek, Kirk Staschke, Luigi Fontana, Roberto Pili. Alternate-day fasting enhances the activity of anti-androgen therapy in prostate cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4239.
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