Assessment of real time internalization of cell penetrating peptide cargos with adaptors
Disciplines
Biochemistry | Molecular Biology
Abstract (300 words maximum)
Cell penetrating peptides are a promising tool for an efficient and effective way to deliver therapeutic cargos to intracellular targets. CPPs have shown the ability to easily permeate cell membranes but are often entrapped in endosomes where they are either recycled out of the cell or digested in lysosomes. The rate of CPP internalization, effect of internalization on target cells, and method of transport across the membrane are poorly understood aspects of CPP delivery. One promising CPP adaptor is TAT-CaM, which consists of HIV transactivator of transcription peptide, the CPP, fused with human calmodulin. Cargo proteins are noncovalently and reversibly linked to TAT-CaM via binding to a calmodulin binding site engineered onto the cargo. During endocytosis and cellular trafficking cargos readily dissociate and efficiently avoid endosomal entrapment. In this study we assess the process of intracellular delivery, endosomal entrapment, and endosomal escape of fluorescent CPP cargos with a variety of adaptors, and how this effects the action of the CPP complex within the cell. We will use confocal microscopy to visualize the process of intracellular delivery of the CPP complex. Assessing this process in real time answers many valuable questions such as cellular responses to CPP internalization, internalization rate, efficiency of cargo dissociation from TAT-CaM and other cargos, rate of endosomal escape in relation to different CPPs, and potential mechanisms of intracellular transport. Answering these questions opens the door to developing more efficient and effective CPP adaptors to deliver therapeutic cargos to intracellular targets.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Jonathan McMurry
Assessment of real time internalization of cell penetrating peptide cargos with adaptors
Cell penetrating peptides are a promising tool for an efficient and effective way to deliver therapeutic cargos to intracellular targets. CPPs have shown the ability to easily permeate cell membranes but are often entrapped in endosomes where they are either recycled out of the cell or digested in lysosomes. The rate of CPP internalization, effect of internalization on target cells, and method of transport across the membrane are poorly understood aspects of CPP delivery. One promising CPP adaptor is TAT-CaM, which consists of HIV transactivator of transcription peptide, the CPP, fused with human calmodulin. Cargo proteins are noncovalently and reversibly linked to TAT-CaM via binding to a calmodulin binding site engineered onto the cargo. During endocytosis and cellular trafficking cargos readily dissociate and efficiently avoid endosomal entrapment. In this study we assess the process of intracellular delivery, endosomal entrapment, and endosomal escape of fluorescent CPP cargos with a variety of adaptors, and how this effects the action of the CPP complex within the cell. We will use confocal microscopy to visualize the process of intracellular delivery of the CPP complex. Assessing this process in real time answers many valuable questions such as cellular responses to CPP internalization, internalization rate, efficiency of cargo dissociation from TAT-CaM and other cargos, rate of endosomal escape in relation to different CPPs, and potential mechanisms of intracellular transport. Answering these questions opens the door to developing more efficient and effective CPP adaptors to deliver therapeutic cargos to intracellular targets.