Antennapedia Homeodomain Research Articles

Determination of Penetratin Secondary Structure in Live Cells with Raman Microscopy

Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm(-1), thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and beta-strand in the cytoplasm, and possibly assembling as beta-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably nonendocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.

Gene delivery using a derivative of the protein transduction domain peptide, K-Antp

Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third α-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide–DNA complex. An optimal K-Antp/DNA formula was nearly 400–600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.

Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila melanogaster

The phenomenon of protein transduction represents internalization of short peptides known as protein transduction domains (PTD) by cells. It is widely used in the development of new preparations for treatment of various brain disorders. However, the drug discovery process is limited by lack of simple and reliable models of blood brain barrier (BBB). These models should meet two main criteria: they should be applicable for testing of large numbers of samples simultaneously reproduce the physiological and functional characteristics of mammalian (including) human BBB. The major goal of this study was to estimate the BBB-crossing ability of known PTD-peptides using Drosophila melanogaster BBB as the model. We demonstrate here that after abdominal administration the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein can cross Drosophila and deliver the apoE mimetic peptide exhibiting neuroprotective properties.

Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

Cellular uptake of cationic gadolinium-DOTA peptide conjugates with and without N-terminal myristoylation

Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding approximately 2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further approximately 40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.

A Novel Cell-penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids

Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.

Driving Forces in the Delivery of Penetratin Conjugated G Protein Fragment

A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.

Cutting Edge: Differential Inhibition of TLR Signaling Pathways by Cell-Permeable Peptides Representing BB Loops of TLRs

We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-kappaB translocation and early IL-1beta mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys(4)-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-kappaB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins.

DNA Recognition by the Brinker Repressor – An Extreme Case of Coupling Between Binding and Folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix α3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the “coupling of binding and folding” phenomenon.

La transduction protéique, de la technologie à la physiologie

In the early 90s, we found that the DNA-binding domain (homeodomain) of Antennapedia, a homeoprotein transcription factor, was internalized by live cells gaining access to their cytoplasm and nuclei. It was soon revealed that internalization is due to the third helix of the homeodomain, composed of sixteen amino acids. This short peptide baptized Penetratin is the first of a large series of transduction peptides widely used for the internalization of all sorts of cargoes in vitro and in vivo. Although transduction peptides are being developed with the latter practical goal, the most intriguing outcome of our initial observation is that full-length homeoproteins are transferred between cells and have non-cell autonomous transcriptional and translational activities. This new signaling mechanism requires that homeoproteins be internalized and secreted. Secretion is Golgi independent and requires a small sequence also present in the homeodomain but distinct from the Penetratin sequence. The consequences of this novel signaling mechanism are briefly discussed.

In vivo delivery of a XIAP (BIR3-RING) fusion protein containing the protein transduction domain protects against neuronal death induced by seizures

The prevention of cell apoptosis is a promising strategy for neuroprotection against brain injury in seizures. X-linked inhibitor of apoptosis protein (XIAP) is regarded as the most potent inhibitor of cell apoptosis. In the present study, we fused the protein transduction domain (PTD) of Antennapedia Homeodomain of Drosophila (AntpHD) to XIAP (BIR3-RING) and explored the neuroprotective effect of XIAP in rats with seizures induced by kainic acid (KA). KA triggered neuronal death in the ipsilateral CA3 subfield of the hippocampus and activation of caspase-3 and -9. PTD-XIAP fusion protein can be delivered into cos7 cells in vitro. We used intraperitoneal injection to deliver the PTD-XIAP fusion protein which can enter into brain, significantly decrease the TUNEL positive cells and increase the number of surviving cells in the ipsilateral CA3 subfield of the hippocampus at 24 h after KA-induced seizures. Furthermore, PTD-XIAP fusion protein attenuated activated caspase-3 and -9. These results demonstrate the neuroprotective effect of PTD-XIAP fusion protein against brain injury possibly through the inhibition of caspase. The significance of these findings in the treatment of epilepsy still needs to be extensively studied.

Structure of Homeodomain-Leucine Zipper/DNA Complexes Studied Using Hydroxyl Radical Cleavage of DNA and Methylation Interference

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.

Apoptosis inhibition in ischemic brain by intraperitoneal PTD-BIR3-RING (XIAP)

Anti-apoptotic treatment is a promising strategy for neuroprotection against various brain injuries resulting from ischemia or neuron degeneration. X-linked inhibitor of apoptosis protein (XIAP) is regarded as the most effective apoptosis inhibitor, in which C-terminal structure BIR3-RING mainly inhibits caspase-9-dependent apoptosis. In the present study, we fused XIAP (BIR3-RING) to the protein transduction domain (PTD) of antennapedia homeodomain of Drosophila (Antp HD), and then used the oxygen glucose deprivation (OGD)-induced hippocampal slices injury in vitro, and the rat transient middle cerebral artery ischemia (tMCAO) models in vivo, to explore the anti-apoptotic effect of this recombinant protein. The results showed that the PTD could efficiently mediate the transduction of BIR3-RING into the hippocampal slices and rat brains. PTD-BIR3-RING could decrease OGD-induced cell death in brain slices ( p < 0.05). Intraperitoneal injection of PTD-BIR3-RING could attenuate terminal deoynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive cells and decrease cleaved caspase-3 in the ischemic bounder zone compared with the control animals ( p < 0.05). Further studies showed that ischemia-induced neurological outcomes were improved in rats with PTD-BIR3-RING treatment ( p < 0.05). These results demonstrate that PTD-BIR3-RING could attenuate cell death in OGD hippocampal slices and decrease cell apoptosis in tMCAO brains through inhibiting of caspase-3 cleavage, suggesting that PTD-mediated protein transduction provides a novel and effective approach for the therapies of brain diseases such as cerebral ischemia.

Present and future of cell-penetrating peptide mediated delivery systems: “Is the Trojan horse too wild to go only to Troy?”

During the last decade, small peptides (10 to 15 amino acids) derived from the HIV-1 Tat protein and from the drosophila Antennapedia homeodomain have been used to internalize various types of molecules into the cells. The way these peptides enter cells is still under investigation and the object of strong controversy. The main discussions rely on whether these peptides are internalized or not in an energy-independent fashion, and, depending on the situation, whether they follow one pathway instead of another. At present, we find in the literature a very large number of data with, at times, some contradictory results. Indeed the diversity of employed peptide sequences, the cell type used, the attachment or not of a cargo molecule, the chemical nature of this cargo itself, and the followed protocol during the experimental process do not simplify the comparison and hence final conclusions about the mechanism of cell entry. However, one common feature emerges with these cell-penetrating peptides: most of them do not show any cell specificity. Despite their demonstrated efficiency in delivering biologically active molecules in in vitro experiments, their use for a therapeutic application in vivo has been the object of a relatively little number of studies, probably because of the quite important amounts of CPP–cargo that needs to be prepared for an accurate and complete in vivo study, but more likely, because of the massive spreading of the cargo all around the body. However, it appears from recent studies that an increased targeting ability of these CPPs is possible, making the use of CPP mediated delivery compatible with an in vivo therapeutic approach.

The Role of Protein-tyrosine Phosphatase 1B in Integrin Signaling

Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading and migration to fibronectin, increases Tyr(527) phosphorylation in Src, and decreases phosphorylation of FAK, p130(Cas), and ERK1/2. In addition, PTP1B "substrate-trapping" mutants bind Tyr(527)-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr(527) and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130(Cas), and that the proline-rich motif PPRPPK (residues 309-314) in PTP1B is essential for the complex formation. We suggest that the specificity of PTP1B for Src pTyr(527) is mediated by protein-protein interactions involving the docking protein p130(Cas) with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr(527) motif.

Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction

TLRs sense pathogens and transmit intracellular signals via the use of specific adapter proteins. We designed a set of "blocking peptides" (BPs) comprised of the 14 aa that correspond to the sequences of the BB loops of the four known Toll-IL-1 resistance (TIR) domain-containing adapter proteins (i.e., MyD88, TIR domain-containing adapter inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and TIR-domain containing adapter protein (TIRAP)) linked to the cell-penetrating segment of the antennapedia homeodomain. LPS (TLR4)-mediated gene expression, as well as MAPK and transcription factor activation associated with both MyD88-dependent and -independent signaling pathways, were disrupted by all four BPs (TRAM approximately MyD88 > TRIF > TIRAP), but not by a control peptide. In contrast, none of the BPs inhibited TLR2-mediated activation of MAPKs. Only the MyD88 BP significantly blocked Pam3Cys-induced IL-1beta mRNA; however, the inhibitory effect was much less than observed for LPS. Our data suggest that the interactions required for a fully functional TLR4 signaling "platform" are disrupted by these BPs, and that the adapter BB loops may serve distinct roles in TLR4 and TLR2 signalosome assembly.

Biological evaluation of penetration domain and killing domain peptides

Cancer gene therapy must impact the majority of cells to be effective. Current gene delivery systems are unable to achieve sufficient transfer efficiency to the tumor cells. Cell killing can be dramatically increased through a bystander effect. Modeling the gene product with synthetic peptides can identify key elements for creating cell killing through a bystander effect. Fluorescent labeled peptides were used for uptake kinetic studies and determination of intracellular localization in human glioblastoma cell lines, rat glioma cells lines and pressurized rat cerebral arteries. The degree of cell killing was assayed using propidium iodide coupled with fluorescence-activated cell sorting (FACS) analysis. Peptides derived from HIV Tat and Drosophila antennapedia homeodomain were taken up by all tumor and primary cells. Attachment of an Mdm-2-binding domain derived from P14(ARF) resulted in cell killing and was independent of domain orientation. Uptake kinetics showed rapid uptake for both tumor and primary cells equilibrating with the external media within 10 min. Intraluminal or extraluminal administration of peptides into pressurized cerebral arteries showed a lack of extravasation across the subbasement lamina. Assay of biological activity following intraluminal administration showed selective suppression of response to vasodilation with no effect on response by smooth muscle cells. The results from these studies identified: (1) a cell trafficking domain and a cytotoxic domain for killing brain tumor cells; (2) that cell killing was independent of the domain orientations with regard to the cell trafficking domain being at the C-terminus or N-terminus; and (3) that the dual domain peptide can also be taken up by endothelial cells as shown by the cerebral artery studies. Hence, localized expression of the cytotoxic gene has the potential to not only kill brain tumor cells, but also tumor endothelium, thus further increasing the effectiveness of the therapy.

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.

PKC? inhibits the hyperglycemia-induced apoptosis signal in adult rat ventricular myocytes

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC betaI/betaII, zeta and epsilon) or activate (PKCepsilon). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC betaI/betaII and zeta blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKCepsilon (epsilonV1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKCepsilon translocation activator (psi epsilonRACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKCepsilon in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKCepsilon protects ARVM from the hyperglycemia-induced death signal.