Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.
Mutations in the gene tafazzin (TAZ) result in Barth syndrome (BTHS). Patients present with hypotonia, cyclic neutropenia, 3-methyglutaconic aciduria, and cardiomyopathy, which is the major cause of mortality. The recessive, X-linked TAZ gene encodes a mitochondrial membrane-associated phospholipid modifying enzyme, which adds unsaturated fatty acid species to monolysocardiolipin to generate mature cardiolipin in the mitochondrial membrane that is essential for mitochondrial morphology and function. To identify intrinsic mitochondrial localization sequences in the human TAZ protein, we made sequential TAZ peptide-eGFP fusion protein expression constructs and analyzed the localization of eGFP fluorescence by confocal microscopy. We assessed these fusion proteins for mitochondrial localization through cotransfection of H9c2 cells with plasmids encoding organellar markers linked to TdTomato. We have identified two peptides of TAZ that are independently responsible for mitochondrial localization. Using CRISPR-generated TAZ knock out cell lines, we found that these peptides are able to direct proteins to mitochondria in the absence of endogenous TAZ. These peptides are not located within the predicted enzymatic clefts of TAZ, implying that some BTHS disease causing mutations may affect mitochondrial localization without affecting transacylase activity. These novel peptides improve our understanding of TAZ intracellular trafficking, provide insight into the molecular basis of BTHS and provide molecular reagents for developing targeted mitochondrial therapies.
The technique of Cre-mediated DNA recombination at loxP sites has been used widely in manipulation of the genome in cultured cells and in living organisms. Local delivery of Cre recombinase protein tagged with a cell-penetrating (or permeable) peptide (Cre-CPP) has the advantage of additional spatial and temporal control when compared to genetic delivery methods. In this chapter, we describe protocols for injection-based intramuscular delivery of Cre-CPP dissolved in hydrogel to skeletal muscle and by ultrasound-guided injection to cardiac muscle in mice.
MHC class I “single-chain trimer” molecules, coupling MHC heavy chain, β2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.
Barth syndrome (BTHS) is an X-linked recessive disease where patients most commonly die from cardiomyopathy-induced heart failure before middle age. BTHS is caused by mutations in the tafazzin (TAZ) gene, resulting in defective TAZ protein. TAZ is an enzyme that generates mature cardiolipin (CL) from monolysocardiolipin (MLCL) in the mitochondrial membrane, a reaction essential for normal mitochondrial function. Current therapies can only treat the symptoms of BTHS. In this study, we propose an enzyme replacement therapy for BTHS which utilizes recombinant human TAZ fused to a cell penetrating peptide (hTAZ-CTP) to facilitate tissue uptake. The efficacy of this protein was tested in vitro on C2C12 TAZ-knockout (TAZ-KO) skeletal myoblasts and in vivo on a myocardial-specific TAZ conditional knockout mouse, modelling the cardiomyopathy associated with BTHS. In vitro tests of TAZ-KO cells, using oxygen consumption rate as a measure of mitochondrial activity, showed treatment of the cells with hTAZ-CTP effected a partial rescue of the fatty acid oxidation capabilities of the TAZ-KO cells. In vivo tests showed that BTHS mice display increasing septal wall thickness over time, an effect halted upon treatment with hTAZ-CTP. Pressure-volume (PV) loop analysis indicated that heart function, impaired in the vehicle-treated BTHS mouse, was similar between treated mice and normal mice. The ratio of MLCL/CL, a direct measure of TAZ enzymatic activity, was measured in heart mitochondria isolated from BTHS and control mice after treatment. The vehicle treated BTHS mouse showed the high MLCL/CL ratio typical of BTHS patients, whereas the MLCL/CL ratios in protein-treated mice matched the much lower ratio of the control mice. Similarly, oxygen consumption rate measurements of these isolated heart mitochondria demonstrated partial rescue by hTAZ-CTP treatment. Coupled with the lack of toxicity observed in the liver, spleen, kidney, and heart due to hTAZ-CTP injection, these results indicate that TAZ enzyme replacement therapy has great potential as a future treatment for BTHS.
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