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Chemical control of protein stability and function in living mice.

ABSTRACT: Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

SUBMITTER: Banaszynski LA

PROVIDER: S-EPMC2605277 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Chemical control of protein stability and function in living mice.

Banaszynski Laura A LA Sellmyer Mark A MA Contag Christopher H CH Wandless Thomas J TJ Thorne Steve H SH

Nature medicine 20080928 10

Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of sec ...[more]

PMID: 18836461

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Project description:BackgroundCytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.ResultsThree different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.ConclusionThe results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

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Chemical control of protein stability and function in living mice.

Publications

Chemical control of protein stability and function in living mice.

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