Bioengineering / Biyomühendislik
Permanent URI for this collectionhttps://hdl.handle.net/11147/4529
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Article Citation - WoS: 7Citation - Scopus: 7Potent Telomerase Activators From a Novel Sapogenin Via Biotransformation Utilizing Camarosporium Laburnicola, an Endophytic Fungus(BioMed Central Ltd., 2023) Küçüksolak, Melis; Yılmaz, Sinem; Ballar Kırmızıbayrak, Petek; Bedir, ErdalBACKGROUND: Cycloartane-type triterpenoids possess important biological activities, including immunostimulant, wound healing, and telomerase activation. Biotransformation is one of the derivatization strategies of natural products to improve their bioactivities. Endophytic fungi have attracted attention in biotransformation studies because of their ability to perform modifications in complex structures with a high degree of stereospecificity. RESULTS: This study focuses on biotransformation studies on cyclocephagenol (1), a novel cycloartane-type sapogenin from Astragalus species, and its 12-hydroxy derivatives (2 and 3) to obtain new telomerase activators. Since the hTERT protein levels of cyclocephagenol (1) and its 12-hydroxy derivatives (2 and 3) on HEKn cells were found to be notable, biotransformation studies were carried out on cyclocephagenol and its 12-hydroxy derivatives using Camarosporium laburnicola, an endophytic fungus isolated from Astragalus angustifolius. Later, immunoblotting and PCR-based ELISA assay were used to screen starting compounds and biotransformation products for their effects on hTERT protein levels and telomerase activation. All compounds showed improved telomerase activation compared to the control group. CONCLUSIONS: As a result of biotransformation studies, seven new metabolites were obtained and characterized, verifying the potential of C. laburnicola as a biocatalyst. Additionally, the bioactivity results showed that this endophytic biocatalyst is unique in transforming the metabolites of its host to afford potent telomerase activators. © 2023. The Author(s).Article Citation - WoS: 8Citation - Scopus: 9Neuroprotective Metabolites Via Fungal Biotransformation of a Novel Sapogenin, Cyclocephagenol(Nature Research, 2022) Küçüksolak, Melis; Üner, Göklem; Ballar Kırmızıbayrak, Petek; Bedir, ErdalCyclocephagenol (1), a novel cycloartane-type sapogenin with tetrahydropyran unit, is only encountered in Astragalus species. This rare sapogenin has never been a topic of biological activity or modification studies. The objectives of this study were; (i) to perform microbial transformation studies on cyclocephagenol (1) using Astragalus endophyte, Alternaria eureka 1E1BL1, followed by isolation and structural characterization of the metabolites; (ii) to investigate neuroprotective activities of the metabolites; (iii) to understand structure–activity relationships towards neuroprotection. The microbial transformation of cyclocephagenol (1) using Alternaria eureka resulted in the production of twenty-one (2–22) previously undescribed metabolites. Oxidation, monooxygenation, dehydration, methyl migration, epoxidation, and ring expansion reactions were observed on the triterpenoid skeleton. Structures of the compounds were established by 1D-, 2D-NMR, and HR-MS analyses. The neuroprotective activities of metabolites and parent compound (1) were evaluated against H2O2-induced cell injury. The structure–activity relationship (SAR) was established, and the results revealed that 1 and several other metabolites had potent neuroprotective activity. Further studies revealed that selected compounds reduced the amount of ROS and preserved the integrity of the mitochondrial membrane. This is the first report of microbial transformation of cyclocephagenol.Article Citation - WoS: 2Citation - Scopus: 3Non-Apoptotic Cell Death Induction Via Sapogenin Based Supramolecular Particles(Nature Publishing Group, 2022) Üner, Göklem; Bedir, Erdal; Serçinoğlu, Onur; Ballar Kırmızıbayrak, PetekThe discovery of novel chemotherapeutics that act through different mechanisms is critical for dealing with tumor heterogeneity and therapeutic resistance. We previously reported a saponin analog (AG-08) that induces non-canonical necrotic cell death and is auspicious for cancer therapy. Here, we describe that the key element in triggering this unique cell death mechanism of AG-08 is its ability to form supramolecular particles. These self-assembled particles are internalized via a different endocytosis pathway than those previously described. Microarray analysis suggested that AG-08 supramolecular structures affect several cell signaling pathways, including unfolded protein response, immune response, and oxidative stress. Finally, through investigation of its 18 analogs, we further determined the structural features required for the formation of particulate structures and the stimulation of the unprecedented cell death mechanism of AG-08. The unique results of AG-08 indicated that supramolecular assemblies of small molecules are promising for the field of anticancer drug development, although they have widely been accepted as nuisance in drug discovery studies.Article Citation - WoS: 13Citation - Scopus: 15The Role of Cycloastragenol at the Intersection of Nrf2/Are, Telomerase, and Proteasome Activity(Elsevier, 2022) Yılmaz, Sinem; Bedir, Erdal; Ballar Kırmızıbayrak, PetekAging is well-characterized by the gradual decline of cellular functionality. As redox balance, proteostasis, and telomerase systems have been found to be associated with aging and age-related diseases, targeting these systems with small compounds has been considered a promising therapeutic approach. Cycloastragenol (CA), a small molecule telomerase activator obtained from Astragalus species, has been reported to positively affect several age-related pathophysiologies, but the mechanisms underlying CA activity have yet to be reported. Here, we presented that CA increased NRF2 nuclear localization and activity leading to upregulation of cytoprotective enzymes and attenuation of oxidative stress-induced ROS levels. Furthermore, CA-mediated induction of telomerase activity was found to be regulated by NRF2. CA not only increased the expression of hTERT but also its nuclear localization via upregulating the Hsp90-chaperon complex. In addition to modulating nuclear hTERT levels at unstressed conditions, CA alleviated oxidative stress-induced mitochondrial hTERT levels while increasing nuclear hTERT levels. Concomitantly, H2O2-induced mitochondrial ROS level was found to be significantly decreased by CA administration. Our data also revealed that CA strongly enhanced proteasome activity and assembly. More importantly, the proteasome activator effect of CA is dependent on the induction of telomerase activity, which is mediated by NRF2 system. In conclusion, our results not only revealed the cross-talk among NRF2, telomerase, and proteasome systems but also that CA functions at the intersection of these three major aging-related cellular pathways.Article Citation - WoS: 7Citation - Scopus: 7Novel Regulation Mechanism of Adrenal Cortisol and Dhea Biosynthesis Via the Endogen Erad Inhibitor Small Vcp-Interacting Protein(Nature Publishing Group, 2022) İlhan, Recep; Üner, Göklem; Yılmaz, Sinem; Atalay Sahar, Esra; Çaylı, Sevil; Erzurumlu, Yalçın; Gözen, Oğuz; Ballar Kırmızıbayrak, PetekEndoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.Article Citation - WoS: 5Citation - Scopus: 6Undescribed Polyether Ionophores From Streptomyces Cacaoi and Their Antibacterial and Antiproliferative Activities(Elsevier, 2022) Gezer, Emre; Üner, Göklem; Küçüksolak, Melis; Kurt, Mustafa Ünver; Doğan, Gamze; Ballar Kırmızıbayrak, Petek; Bedir, ErdalPolyether ionophores represent a large group of naturally occurring compounds mainly produced by Streptomyces species. With previously proven varieties of bioactivity including antibacterial, antifungal, antiparasitic, antiviral and anti-tumor effects, the discovery of undescribed polyethers leading to development of efficient therapeutics has become important. As part of our research on polyether-rich Streptomyces cacaoi, we previously performed modification studies on fermentation conditions to induce synthesis of specialized metabolites. Here, we report four undescribed and nine known polyether compounds from S. cacaoi grown in optimized conditions. Antimicrobial activity assays revealed that four compounds, including the undescribed (6), showed strong inhibitory effects over both Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA) growth. Additionally, K41-A and its C15-demethoxy derivative exhibited significant cytotoxicity. These results signified that selectivity of C15-demethoxy K41-A towards cancer cells was higher than K41-A, which prompted us to conduct mechanistic experiments. These studies showed that this uninvestigated compound acts as a multitarget compound by inhibiting autophagic flux, inducing reactive oxygen species formation, abolishing proteasome activity, and stimulating ER stress. Consequently, the optimized fermentation conditions of S. cacaoi led to the isolation of undescribed and known polyethers displaying promising activities.Article Citation - WoS: 19Citation - Scopus: 21Telomerase Activators From 20(27)-Octanor Via Biotransformation by the Fungal Endophytes(Academic Press, 2021) Duman, Seda; Ekiz, Güner; Yılmaz, Sinem; Yusufoğlu, Hasan; Ballar Kırmızıbayrak, Petek; Bedir, ErdalCycloastragenol [20(R),24(S)-epoxy-3 beta,6 alpha,16 beta,25-tetrahydroxycycloartane] (CA), the principle sapogenol of many cycloartane-type glycosides found in Astragalus genus, is currently the only natural product in the anti-aging market as telomerase activator. Here, we report biotransformation of 20(27)-octanor-cycloastragenol (1), a thermal degradation product of CA, using Astragalus species originated endophytic fungi, viz. Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae and Camarosporium laburnicola. Fifteen new biotransformation products (2-16) were isolated, and their structures were established by NMR and HRESIMS. Endophytic fungi were found to be capable of performing hydroxylation, oxidation, ring cleavage-methyl migration, dehydrogenation and Baeyer-Villiger type oxidation reactions on the starting compound (1), which would be difficult to achieve by conventional synthetic methods. In addition, the ability of the metabolites to increase telomerase activation in Hekn cells was evaluated, which showed from 1.08 to 12.4-fold activation compared to the control cells treated with DMSO. Among the compounds tested, 10, 11 and 12 were found to be the most potent in terms of telomerase activation with 12.40-, 7.89- and 5.43-fold increase, respectively (at 0.1, 2 and 10 nM concentrations, respectively).Article Citation - WoS: 4Citation - Scopus: 5Identification of a Noncanonical Necrotic Cell Death Triggered Via Enhanced Proteolysis by a Novel Sapogenol Derivative(American Chemical Society, 2020) Üner, Göklem; Tağ, Özgür; Erzurumlu, Yalçın; Ballar Kırmızıbayrak, Petek; Bedir, ErdalSmall molecules which activate distinct cell death pathways have promising high potential for anticancer drug research. Especially, regulated necrosis draws attention as an alternative cell death mechanism to overcome the drug resistance. Here, we report that a new semisynthetic saponin analogue (AG-08) triggers necrotic cell death with unprecedented pathways. AG-08-mediated necrosis depends on enhanced global proteolysis involving calpains, cathepsins, and caspases. Moreover, AG-08 generates several alterations in lysosomal function and physiology including membrane permeabilization, redistribution toward the perinuclear area, and lastly excessive tubulation. As a consequence of lysosomal impairment, the autophagic process was abolished via AG-08 treatment. Collectively, in addition to its ability to induce necrotic cell death, which makes AG-08 a promising candidate to cope with drug resistance, its unique activity mechanisms including autophagy/lysosome impairment and enhancement of proteolysis leading a strong death capacity emphasizes its potential for anticancer drug research. ©Article Citation - WoS: 24Citation - Scopus: 23Polyethers Isolated From the Marine Actinobacterium Streptomyces Cacaoi Inhibit Autophagy and Induce Apoptosis in Cancer Cells(Elsevier, 2019) Khan, Nasar; Yılmaz, Sinem; Aksoy, Semiha; Uzel, Ataç; Tosun, Çiğdem; Ballar Kırmızıbayrak, Petek; Bedir, ErdalPolyether compounds, a large group of biologically active metabolites produced by Streptomyces species have been reported to show a variety of bioactivity such as antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Since some of these compounds target cancer stem cells and multi-drug resistant cancer cells, this family of compounds have become of high interest. In this study, three polyether-type metabolites (1-3), one of which was a new natural product (3), were isolated from the marine derived Streptomyces cacaoi via antimicrobial activity-guided fractionation studies. As several polyether compounds with structural similarity such as monensin have been linked with autophagy and cell death, we first assessed the cytotoxicity of these three compounds. Compounds 2 and 3, but not 1, were found to be cytotoxic in several cell lines with a higher potency towards cancer cells. Furthermore, 2 and 3 caused accumulation of both autophagy flux markers LC3-II and p62 along with cleavage of caspase-3, caspase-9 and poly (ADP-ribose) polymerase 1 (PARP-1). Interestingly, prolonged treatment of the compounds caused a dramatic downregulation of the proteins related to autophagasome formation in a dose dependent manner. Our findings provide insights on the molecular mechanisms of the polyether-type polyketides, and signify their potency as chemotherapeutic agents through inhibiting autophagy and inducing apoptosis.Article Citation - WoS: 23Citation - Scopus: 22Microbial Transformation of Cycloastragenol and Astragenol by Endophytic Fungi Isolated From Astragalus Species(American Chemical Society, 2019) Ekiz, Güner; Yılmaz, Sinem; Yusufoğlu, Hasan; Ballar Kırmızıbayrak, Petek; Bedir, ErdalBiotransformation of Astragalus sapogenins (cycloastragenol (1) and astragenol (2)) by Astragalus species originated endophytic fungi resulted in the production of five new metabolites (3, 7, 10, 12, 14) together with 10 known compounds. The structures of the new compounds were established by NMR spectroscopic and HRMS analysis. Oxygenation, oxidation, epoxidation, dehydrogenation, and ring cleavage reactions were observed on the cycloartane (9,19-cyclolanostane) nucleus. The ability of the compounds to increase telomerase activity in neonatal cells was also evaluated. After prescreening studies to define potent telomerase activators, four compounds were selected for subsequent bioassays. These were performed using very low doses ranging from 0.1 to 30 nM compared to the control cells treated with DMSO. The positive control cycloastragenol and 8 were found to be the most active compounds, with 5.2- (2 nM) and 5.1- (0.5 nM) fold activations versus DMSO, respectively. At the lowest dose of 0.1 nM, compounds 4 and 13 provided 3.5- and 3.8-fold activations, respectively, while cycloastragenol showed a limited activation (1.5-fold).