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Item Genome of Pythium myriotylum Uncovers an Extensive Arsenal of Virulence-Related Genes among the Broad-Host-Range Necrotrophic Pythium Plant Pathogens(AMER SOC MICROBIOLOGY, 2022-01-01) Daly, Paul; Zhou, Dongmei; Shen, Danyu; Chen, Yifan; Xue, Taiqiang; Chen, Siqiao; Zhang, Qimeng; Zhang, Jinfeng; McGowan, Jamie; Cai, Feng; Pang, Guan; Wang, Nan; Sheikh, Taha Majid Mahmood; Deng, Sheng; Li, Jingjing; Soykam, Huseyin Okan; Kara, Irem; Fitzpatrick, David A.; Druzhinina, Irina S.; Akcapinar, Gunseli Bayram; Wei, LihuiThe Pythium (Peronosporales, Oomycota) genus includes devastating plant pathogens that cause widespread diseases and severe crop losses. Here, we have uncovered a far greater arsenal of virulence factor-related genes in the necrotrophic Pythium myriotylum than in other Pythium plant pathogens. The genome of a plant-virulent P. myriotylum strain (similar to 70 Mb and 19,878 genes) isolated from a diseased rhizome of ginger (Zingiber officinale) encodes the largest repertoire of putative effectors, proteases, and plant cell wall-degrading enzymes (PCWDEs) among the studied species. P. myriotylum has twice as many predicted secreted proteins than any other Pythium plant pathogen. Arrays of tandem duplications appear to be a key factor of the enrichment of the virulence factor-related genes in P. myriotylum. The transcriptomic analysis performed on two P. myriotylum isolates infecting ginger leaves showed that proteases were a major part of the upregulated genes along with PCWDEs, Nep1-like proteins (NLPs), and elicitin-like proteins. A subset of P. myriotylum NLPs were analyzed and found to have necrosis-inducing ability from agroinfiltration of tobacco (Nicotiana benthamiana) leaves. One of the heterologously produced infection-upregulated putative cutinases found in a tandem array showed esterase activity with preferences for longer-chain-length substrates and neutral to alkaline pH levels. Our results allow the development of science-based targets for the management of P. myriotylum-caused disease, as insights from the genome and transcriptome show that gene expansion of virulence factor-related genes play a bigger role in the plant parasitism of Pythium spp. than previously thought. IMPORTANCE Pythium species are oomycetes, an evolutionarily distinct group of filamentous fungus-like stramenopiles. The Pythium genus includes several pathogens of important crop species, e.g., the spice ginger. Analysis of our genome from the plant pathogen Pythium myriotylum uncovered a far larger arsenal of virulence factor-related genes than found in other Pythium plant pathogens, and these genes contribute to the infection of the plant host. The increase in the number of virulence factor-related genes appears to have occurred through the mechanism of tandem gene duplication events. Genes from particular virulence factor-related categories that were increased in number and switched on during infection of ginger leaves had their activities tested. These genes have toxic activities toward plant cells or activities to hydrolyze polymeric components of the plant. The research suggests targets to better manage diseases caused by P. myriotylum and prompts renewed attention to the genomics of Pythium plant pathogens.Item COMPARATIVE PHYSIOCHEMICAL ANALYSIS OF HYDROPHOBINS PRODUCED IN ESCHERICHIA COLI AND PICHIA PASTORIS(ELSEVIER, 2017-01-01) Przylucka, Agnes; Akcapinar, Gunseli Bayram; Bonazza, Klaus; Mello-de-Sousa, Thiago M.; Mach-Aigner, Astrid R.; Lobanov, Victor; Grothe, Hinrich; Kubicek, Christian P.; Reimhult, Erik; Druzhinina, Irina S.Hydrophobins (HFBs) are small surface-active proteins secreted by filamentous fungi. Being amphiphilic, they spontaneously form layers that convert surfaces from hydrophilic to hydrophobic and vice versa. We have compared properties of the class II HFB4 and HFB7 from Trichoderma virens as produced in Escherichia coli and Pichiapastoris. Since the production in E. coli required denaturation/renaturation steps because of inclusion bodies, this treatment was also applied to HFBs produced and secreted in yeast. The protein yields for both systems were similar. Both HFBs produced by E. coli proved less active on PET compared to HFBs produced in P. pastoris. HFBs produced in E. coli decreased the hydrophilicity of glass the most, which correlated with the adsorption of a more dense protein layer on glass compared to HFBs produced in P. pastoris. The hydrophobins produced in P. pastoris formed highly structured monolayers. Layers of hydrophobins produced in E. coli were less prone to self-organization. Our data suggests that irrespective of the production host, the HFBs could be used in various applications that are based on their surface activity. However, the production host and the subsequent purification procedure will influence the stability of HFB layers. In the area of high-value biomedical devices and nanomaterials, where the formation of highly ordered protein monolayers is essential, our results point to P. pastoris as the preferred production host. Furthermore, the choice of an appropriate hydrophobin for a given application appears to be equally important. (C) 2017 The Authors. Published by Elsevier B.V.Item Molecular modelling of the FOXO4-TP53 interaction to design senolytic peptides for the elimination of senescent cancer cells(ELSEVIER, 2021-01-01) Le, Hillary H.; Cinaroglu, Suleyman S.; Manalo, Elise C.; Ors, Aysegul; Gomes, Michelle M.; Sahbaz, Burcin Duan; Bonic, Karla; Marmolejo, Carlos A. Origel; Quentel, Arnaud; Plaut, Justin S.; Kawashima, Taryn E.; Ozdemir, E. Sila; Malhotra V, Sanjay; Ahiska, Yavuz; Sezerman, Ugur; Akcapinar, Gunseli Bayram; Saldivar, Joshua C.; Timucin, Emel; Fischer, Jared M.Background: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. Interpretation: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health \& Science University. (C) 2021 The Authors. Published by Elsevier B.V.Item The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores(PUBLIC LIBRARY SCIENCE, 2021-01-01) Cai, Feng; Zhao, Zheng; Gao, Renwei; Chen, Peijie; Ding, Mingyue; Jiang, Siqi; Fu, Zhifei; Xu, Pingyong; Chenthamara, Komal; Shen, Qirong; Akcapinar, Gunseli Bayram; Druzhinina, Irina S.Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins-hydrophobins (HFBs)- that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.Item Modulating the thermostability of Endoglucanase I from Trichoderma reesei using computational approaches(OXFORD UNIV PRESS, 2015-01-01) Akcapinar, Gunseli Bayram; Venturini, Alessandro; Martelli, Pier Luigi; Casadio, Rita; Sezerman, Ugur O.In the last decades, effective cellulose degradation became a major point of interest due to the properties of cellulose as a renewable energy source and the widespread application of cellulases (the cellulose degrading enzymes) in many industrial processes. Effective bioconversion of lignocellulosic biomass into soluble sugars for ethanol production requires use of thermostable and highly active cellulases. The library of current cellulases includes enzymes that can work at acidic and neutral pH in a wide temperature range. However, only few cellulases are reported to be thermostable. In order to alleviate this, we have performed a hybrid approach for the thermostabilization of a key cellulase, Endoglucanase I (EGI) from Trichoderma reesei. We combined in silico and in vitro experiments to modulate the thermostability of EGI. Four different predictive algorithms were used to set up a library of mutations. Three thermostabilizer mutations (Q126F, K272F, Q274V) were selected and molecular dynamics simulations at room temperature and high temperatures were performed to analyze the effect of the mutations on enzyme structure and stability. The mutations were then introduced into the endoglucanase 1 gene, using site-directed mutagenesis, and the effect of the mutations on enzyme structure and stability were determined. MD simulations supported the fact that Q126F, K272F and Q274V mutations have a thermostabilizing effect on the protein structure. Experimental studies validated that all of the mutants exhibited higher thermostability compared with native EGI albeit with a decrease in specific activity.Item Computational approaches for de novo design and redesign of metal-binding sites on proteins(PORTLAND PRESS LTD, 2017-01-01) Akcapinar, Gunseli Bayram; Sezerman, Osman UgurMetal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation of metal ions or clusters in proteins. Approximately one-third of these proteins in the databases contain metal ions. Many biological and chemical processes in nature involve metal ion-binding proteins, aka metalloproteins. Many cellular reactions that underpin life require metalloproteins. Most of the remarkable, complex chemical transformations are catalysed by metalloenzymes. Realization of the importance of metal-binding sites in a variety of cellular events led to the advancement of various computational methods for their prediction and characterization. Furthermore, as structural and functional knowledgebase about metalloproteins is expanding with advances in computational and experimental fields, the focus of the research is now shifting towards de novo design and redesign of metalloproteins to extend nature's own diversity beyond its limits. In this review, we will focus on the computational toolbox for prediction of metal ion-binding sites, de novo metalloprotein design and redesign. We will also give examples of tailor-made artificial metalloproteins designed with the computational