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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 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.