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  • Methylated CpGs can recruit transcriptional co repressors to

    2020-11-19

    Methylated CpGs can recruit transcriptional co-repressors to prevent transcription factors (TFs) from promoting gene expression by tightly packing calcium calmodulin dependent protein kinase structures [38]. Two CpG islands in the IPT5b promoter region showed higher methylation level in M9 rootstock compared to Mr (Fig. 3b). However, IPT5b expression was up-regulated in the root when treated with 5-azaC (Fig. 3e), suggesting that IPT5b expression is likely repressed by DNA methylation. Although, IPT5b expression in M9 rootstock increased upon treatment with 5-azaC, the levels were still lower than the Mr rootstock (Fig. 1, Fig. 3e), implying a limitation of the 5-azaC treatment, or there may be other mechanism that regulate IPT5b expression. In conclusion, we found low IPT5b expression and high levels of methylation in the IPT5b promoter region that correlated with low root trans-zeatin biosynthesis in M9 rootstock. Taken together, the methylation and subsequent lower IPT5b expression may induce dwarfing in the M9 rootstock.
    Conflict of interest
    Acknowledgements This project was partially supported by Special Fund for Agro-scientific Research in the Public Interest (201203075), the Earmarked Fund for China Agriculture Research System (CARS-28), National Key Technology Support Program (2013BAD02B01-4) and Beijing Collaborative Innovation Center for Eco-environmental Improvement with Forestry and Fruit Trees (CEFF-PXM2016-014207-000038).
    Introduction Several strategies for targeted cancer therapy involving glucarpidase, also known as Carboxypeptidase G2 (CPG2), have been put into clinical practice in recent years [1]. (Glucarpidase or CPG2 will be used interchangeably throughout the text). Glucarpidase has proved particularly useful in Antibody Directed Enzyme Prodrug Therapy (ADEPT), in which it accumulates at the site of a tumor via a tumor-specific antibody, after that it converts a prodrug into an active drug [[2], [3], [4]]. In contrast, in cases of methotrexate-induced toxicity, glucarpidase is administered to convert this anti-cancer agent to a less harmful compound (4-deoxy-4-amino-N10-methylpteroic acid) that is excreted via a hepatic pathway. The enzyme is typically given in high doses to patients, thereby decreasing the risk of renal failure [[5], [6], [7], [8], [9]]. The re-assortment of mutations to produce favorable combinations that can undergo natural selection is a critical component of biological evolution. This process can be simulated by directed evolution, which has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory uses error-prone DNA replication in vitro to generate genetic diversity and specific screens to identify protein variants with desired properties [10]. Where necessary, the genes for these variants can then be shuffled in a process akin to homologous recombination to achieve further improvements [10]. In several instances, chimeric enzymes with improved activity and stability have been isolated from libraries constructed using DNA shuffling [[11], [12], [13], [14]]. In other cases, the method resulted in libraries with either too many mutations calcium calmodulin dependent protein kinase in each gene [15] or too few crossovers [16] to be useful. DNA shuffling can take advantage of orthologous proteins to repurpose functional diversity from nature, i.e. in addition to using error-prone replication in vitro, it can be used to shuffle distantly related existing sequences to take advantage of the natural diversity that exists within a population and to provide a means to eliminate deleterious mutations that may accumulate in strains [17]. On the other hand, it is limited by the degree of sequence homology shared by the existing sequence variants [10].
    Materials and methods
    Results
    Discussion Glucarpidase, the recombinant form of CPG2, has been used for more than two decades as a detoxifying agent for MTX and also in targeted cancer therapies such as ADEPT. However, its usefulness in both treatment regimens has been limited by its relatively low specific activity and the fact that patients often develop antibodies against it after repeated administration. In our previous study [29], we successfully produced two long-acting variants of glucarpidase, PEGylated glucarpidase and HSA-fused Glucarpidase. We demonstrated that both “biobetter” glucarpidases are less immunogenic and had prolonged half-lives relative to the native enzyme. However, the study did not address the question of the native enzymes relatively low specific activity. In the present work, we used mutagenesis techniques to produce further “biobetter” glucarpidase variants with improved activity.