Intracellular signal transduction is mediated in large part by the interaction(s) and subsequent modification(s), e.g., phosphorylation, of key regulatory proteins. Protein complexes resulting from the juxtaposition of anywhere from just two to as many as a dozen or more proteins control a myriad of cellular functions. In order to further study the interaction(s) of these proteins and their role in regulating cellular function, the use of cell permeable peptides has been employed as a means to introduce into the cell small pieces of these proteins (i.e., peptides) in order to disrupt the interaction of these proteins and study the downstream events.
Small basic (i.e., lysine and/or arginine-rich) peptides derived from the transduction domains of a number of proteins, including the third transmembrane domain of the Antennapedia homeodomain (also known as Penatratin) and the HIV-tat protein, can cross cell membranes without the need for a receptor or other transport carrier. Other mechanisms to introduce peptides into cells include using polyarginine peptides as mimics of the HIV-tat protein, the addition of a fatty acid, such as palmitic or stearic acid or the addition of other hydrophobic groups. The addition of one or more of the above vectors allows certain molecules to become cell-permeable. In addition to modifying peptides, other biologically active molecules such as DNA or proteins can be introduced into cells.
The ultimate destination for this material can be determined through the use of general cell-permeability vectors, such as those above, or through the creation of directed vectors, which might contain for example nuclear localization signals, resulting in the transport of the material of interest to the nucleus. It is always important to keep in mind that design of these molecules requires careful consideration, as not all molecules will in fact be able to enter cells or will be delivered to the intended cellular compartment. It is advisable in some cases to add dyes or other molecules to confirm that the material has been introduced into the cell. In addition, it might be necessary to utilize more than one delivery strategy as well as the use of a scrambled control to rule out nonspecific effects due to the presence of the various components of the system being used.
In cases where the proteins that are interacting is known but the exact site(s) at which the proteins interact is unknown, protein-protein interactions can be studied through the use of zero-length, self-hydrolyzing crosslinkers. In general, a series of overlapping, lower purity peptides can be used first in a broken/permeablized cell system to determine the site(s) of interaction using crosslinkers are introduced to covalently link proteins that are in close proximity. Antibodies or other methods to recover the protein-peptide complex (i.e., biotinylated peptide, epitope-tagged protein) can then be employed to immunoprecipitate protein complexes of interest, and the MS lab at 21st Century Biochemicals can map the site(s) of interaction between the proteins via proteolytic fragment analysis. Once the sites have been identified, suitable cell-permeable peptides can be made to try and disrupt the association between the proteins of interest.
The design of cell-permeable molecules can be achieved in a cost-efficient manner if the projects initial stages are well thought out. The scientists at 21st Century Biochemicals have extensive experience in the design and production of peptides modified with cell-permeabilizing vectors, and can provide expert technical assistance. Together with our experienced mass spectrometry lab and peptide synthesis and purification labs, we are able to provide one stop shopping for your protemics needs.
