Directed evolution and predictive modelling of galactose oxidase towards bulky benzylic and unactivated secondary alcohols

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Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism

FoxO3a knockdown induces, while FoxO1 knockdown inhibits, autophagy.

<p>(A) Immunoblot analysis of lysates from PC3 cells transfected with control siRNA (siLuc) or that targeting FoxO1 (siFoxO1), with or without rapamycin treatment. PC3 cells were transfected with the indicated siRNA for 48 h before subsequent treatment with DMSO, 20 nM or 100 nm rapamycin (Rap) for 24 h prior to harvest and processing. (B) PC3 cells were transfected with the indicated siRNA for 48 h prior to media change, whereupon the cells were subjected to the indicated growth conditions; these conditions are DMEM in the presence (Normal) or absence of 10% FBS (serum -), or in the absence of D-glucose (with 10% FBS), as indicated. Cells were harvested 6 h after exposure to these conditions, and processed for immunoblot analysis of the indicate proteins. (C) Knockdown of FoxO1 with two targeting siRNAs suppresses autophagy in PC3 cells. (D, E, F) illustrate similar study procedures as (A, B, C), but with siRNAs targeting FoxO3a in PC3 cells. All experiments have been performed three times with similar results.</p

FoxO3a and FoxO1 regulation of autophagy is unlikely to be dependent on mTORC1 activity.

<p>(A) PC3 cells were transfected with siRNAs targeting FoxO3a as indicated. Following 48 h of transfection, cells were subjected to rapamycin treatment for 24 h prior to processing for immunoblot analysis of the indicated proteins. (B) PC3 cells underwent similar study as described in (A), except siRNA targeting FoxO1 was used as indicated. All experiments have been performed three times with similar results.</p

FoxO3a knockdown increases autophagic flux.

<p>(A) PC3, MDA-MB-231, and HCT116 cells were transfected with control siRNA (siLuc) or that targeting FoxO3a (siFoxO3a). Subsequently, these cells were exposed to control vehicle or 50 µM chloroquine 72 h post transfection for 3 h before cell lysates preparation for immunoblot analysis of the indicated proteins. (B) Confocal microscopy of MDA-MB231 cell stably expressing tandem fluorescent mRFP-GFP-LC3 following transfection by either control or FoxO3a siRNA. Images were taken 72 h after transfection of the indicated siRNA. (C) Quantitative analysis of the images from the experiment shown in panel B using MetaMorph software to determine the average number of RFP-positive particles per cell in control (siLuc) and siFoxO3a treated cells. (D) Colocalization analysis of RFP and GFP from experiment described in panel B to assess autophagic progression. RFP and GFP colocalization indicated by Pearson Coefficients was analyzed by ImageJ software. In both (C) and (D), >50 cells were analyzed for each condition. Data are presented as Mean ± S.E.M. (“**”, <i>p</i><0.01), and detailed methods are described in Experimental Procedures. (E) PC3 cells were transfected with control siRNA (siLuc) or that targeting Atg5 (siAtg5), FoxO3a (siFoxO3a), or combination of both, as indicated. Cells were harvested 72 h after transfection and the lysates were processed for analysis.</p

FoxO3a negatively regulates autophagy through inhibition of FoxO1 transcription.

<p>(A) PC3 cells were transfected with siRNA for luciferase (siLuc), FoxO1 (siFoxO1), or FoxO3a (siFoxO3a), or combinations, as indicated. Cells were harvested 72 h after transfection, and cell lysates processed for immunoblot analysis of the indicated proteins. (B) FoxO3a knockdown with two FoxO3a targeting siRNA to illustrate the impact on FoxO1 protein level. (C) Real-time PCR analysis of FoxO1 and FoxO3a mRNA levels in control siRNA (gray) or FoxO3a siRNA (black) transfected cells. Cells were harvested and processed 48 h after transfection; 18S ribosomal RNA was analyzed as the normalization control. Data was presented as Mean ± S.D. (“**”, <i>p</i><0.01). (D) PC3 cells were transfected with control siRNA or FoxO3a siRNA as indicated. Following 48 h of transfection, cells were treated with either DMSO control or 10 µg/ml cycloheximide for 24 h as indicated prior to being harvested for immunoblot analysis of the indicated proteins. The FoxO1/GAPDH ratio for each condition is as presented after band quantification by ImageJ. (E) Real-time PCR analysis of endogenous FoxO1 expression level in PC3 cells transfected with either control plasmid (vector) or that expressing FoxO3a(r); in each group of the plasmid transfected cells, either control siRNA or FoxO3a siRNA were concurrently introduced. The cells are harvested for RNA preparation and q-PCR analysis 72 h post transfection; FoxO1 transcript levels were analyzed, as described in Experimental Procedures. 18S was used as the normalization control. Data was presented as Mean ± S.D. (“**”, <i>p</i><0.01). All experiments have been performed three times with similar results.</p

FoxO3a over expression inhibits autophagy.

<p>(A) PC3 cells were transfected with either control siRNA or that targeting FoxO3a; each group of cells was also co-transfected with either ectopic expression vector control or that express FoxO3a(r). The cell lysates were harvested for immunoblot 72 h post transfection. (B) PC3 cells were transfected with 3 different expression plasmids, which are vector-Flag control, Flag-FoxO3a or Flag-FoxO3a-3A, respectively; the cell lysates were prepared 72 h post transfection for immunoblot analysis of the indicated proteins.</p

Directed Evolution and Predictive Modelling of Galactose Oxidase towards Bulky Benzylic and Unactivated Secondary Alcohols

The growth of industrial biocatalysis for sustainable chemical manufacturing has been limited by the narrow range of chemistries associated with natural enzymes and experiment-intensive regimes of enzyme engineering. Consequently, there has been deep interest to expand enzyme substrate scopes for broader synthetic utility, and to streamline the enzyme engineering process. In the field of alcohol oxidation, galactose oxidase (GOase) is one of the most established enzymes capable of this important chemical transformation under benign conditions. However, the applicability of GOase towards more complex molecules such as those frequently found in the pharmaceutical, or agrochemical industries remains restricted. Here, by employing a combined approach of directed evolution and predictive modelling, we have identified new GOases with significantly expanded substrate specificity toward both bulky benzylic and unactivated secondary alcohols, showing activity enhancements of up to 2,400-fold compared to the reported benchmark M3-5 mutant. Beneficial mutations conveying relaxed substrate enantioselectivity biases (R/S ratios down to 1.05) and higher thermostabilities (up to 20-fold versus benchmark) have also been identified. We have developed predictive models based on computational tools YASARA, FoldX, SCWRL and Glide that are well correlated with features related to enzyme structure, selectivity, protein stability and catalytic activity. The generated enzyme activity models based on Glide-MM/GBSA (r = -0.85) and YASARA (r = -0.89) have successfully predicted the activity trend of a family of related substrates based on the 1-phenyl-1-alkyl alcohol scaffold with varying alkyl chain lengths. It is envisioned that these in silico models can serve as valuable tools to explore desirable enzyme characteristics, establish enzyme substrate scopes, and accelerate biocatalyst development, thus promoting it as a competitive and competent solution for sustainable chemical manufacturing