Dataset Information

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

ABSTRACT: Therapeutic proteins are indispensable in treating numerous human diseases. However, therapeutic proteins often suffer short serum half-life. In order to extend the serum half-life, a natural albumin ligand (a fatty acid) has been conjugated to small therapeutic peptides resulting in a prolonged serum half-life via binding to patients' serum albumin in vivo. However, fatty acid-conjugation has limited applicability due to lack of site-specificity resulting in the heterogeneity of conjugated proteins and a significant loss in pharmaceutical activity. In order to address these issues, we exploited the site-specific fatty acid-conjugation to a permissive site of a protein, using copper-catalyzed alkyne-azide cycloaddition, by linking a fatty acid derivative to p-ethynylphenylalanine incorporated into a protein using an engineered pair of yeast tRNA/aminoacyl tRNA synthetase. As a proof-of-concept, we show that single palmitic acid conjugated to superfolder green fluorescent protein (sfGFP) in a site-specific manner enhanced a protein's albumin-binding in vitro about 20 times and the serum half-life in vivo 5 times when compared to those of the unmodified sfGFP. Furthermore, the fatty acid conjugation did not cause a significant reduction in the fluorescence of sfGFP. Therefore, these results clearly indicate that the site-specific fatty acid-conjugation is a very promising strategy to prolong protein serum half-life in vivo without compromising its folded structure and activity.

SUBMITTER: Lim SI

PROVIDER: S-EPMC4437666 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

Lim Sung In SI Mizuta Yukina Y Takasu Akinori A Hahn Young S YS Kim Yong Hwan YH Kwon Inchan I

Journal of controlled release : official journal of the Controlled Release Society 20130602 2

Therapeutic proteins are indispensable in treating numerous human diseases. However, therapeutic proteins often suffer short serum half-life. In order to extend the serum half-life, a natural albumin ligand (a fatty acid) has been conjugated to small therapeutic peptides resulting in a prolonged serum half-life via binding to patients' serum albumin in vivo. However, fatty acid-conjugation has limited applicability due to lack of site-specificity resulting in the heterogeneity of conjugated prot ...[more]

PMID: 23735573

Similar Datasets

Project description:BackgroundAutosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins.ResultsTo test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates.ConclusionsTogether, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

Dataset Information

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

Publications

Site-specific fatty acid-conjugation to prolong protein half-life in vivo.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets