Project Title

Mitochondrial targeting of protein cargos exogenously delivered via a novel cell-penetrating peptide adaptor system

Presenters

Academic department under which the project should be listed

CSM - Molecular and Cellular Biology

Faculty Sponsor Name

Jonathan L. McMurry

Additional Faculty

Julia LeCher, Dept. of Molecular & Cellular Biology, jwand@kennesaw.edu

Abstract (300 words maximum)

Cell penetrating peptides (CPPs) have been used to deliver molecular cargos into a variety of cells and cell types and hold great promise for altering environments within living cells for therapeutic and research purposes, e.g. protein replacement therapy for metabolic diseases. CPPs and biomolecules to which they are attached are rapidly endocytosed, but there is a growing recognition that they become entrapped in endosomes and are eventually targeted for degradation. Our group has developed a CPP-adaptor system that reversibly binds cargo proteins in a Ca2+-dependent manner, solving the so-called ‘endosomal escape problem.’ Ca2+-flux during cellular trafficking induces release of cargos from CPPs, allowing their subsequent delivery to the cytoplasm. The purpose of the present study was to determine if efficient targeting to mitochondria is possible. A synthetic gene encoding a model cargo consisting of myoglobin fused to a His-tag, a calmodulin binding site (CBS) and a mitochondrial targeting sequence (MTS) was made. Protein was expressed and purified from E. coli using metal affinity chromatography. MTS-CBS-myo was spontaneously bound the CPP-adaptor, TAT-CaM. Binding and Ca2+-dependent release was characterized with biolayer interferometry (BLI). Binding was nanomolar in affinity in the presence of Ca2+, and negligible in its absence. MTS-CBS-myo was fluorescently labelled and delivered into baby hamster kidney cells with TAT-CaM. Penetration was assayed by confocal microscopy. Mitochondrial targeting was verified by observing colocalization of MTS-CBS-myo with a fluorescent mitochondrial marker. Future directions include delivering proteins with activities relevant to mitochondrial biology and mitochondrial disease, e.g. superoxide dismutase for alleviation of oxidative stress that leads to mitochondrial DNA mutations.

Project Type

Poster

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Mitochondrial targeting of protein cargos exogenously delivered via a novel cell-penetrating peptide adaptor system

Cell penetrating peptides (CPPs) have been used to deliver molecular cargos into a variety of cells and cell types and hold great promise for altering environments within living cells for therapeutic and research purposes, e.g. protein replacement therapy for metabolic diseases. CPPs and biomolecules to which they are attached are rapidly endocytosed, but there is a growing recognition that they become entrapped in endosomes and are eventually targeted for degradation. Our group has developed a CPP-adaptor system that reversibly binds cargo proteins in a Ca2+-dependent manner, solving the so-called ‘endosomal escape problem.’ Ca2+-flux during cellular trafficking induces release of cargos from CPPs, allowing their subsequent delivery to the cytoplasm. The purpose of the present study was to determine if efficient targeting to mitochondria is possible. A synthetic gene encoding a model cargo consisting of myoglobin fused to a His-tag, a calmodulin binding site (CBS) and a mitochondrial targeting sequence (MTS) was made. Protein was expressed and purified from E. coli using metal affinity chromatography. MTS-CBS-myo was spontaneously bound the CPP-adaptor, TAT-CaM. Binding and Ca2+-dependent release was characterized with biolayer interferometry (BLI). Binding was nanomolar in affinity in the presence of Ca2+, and negligible in its absence. MTS-CBS-myo was fluorescently labelled and delivered into baby hamster kidney cells with TAT-CaM. Penetration was assayed by confocal microscopy. Mitochondrial targeting was verified by observing colocalization of MTS-CBS-myo with a fluorescent mitochondrial marker. Future directions include delivering proteins with activities relevant to mitochondrial biology and mitochondrial disease, e.g. superoxide dismutase for alleviation of oxidative stress that leads to mitochondrial DNA mutations.