Transduction Domain Research Articles

Delivery of transcription factors as modulators of cell differentiation.

Fundamental studies performed during the last decades have shown that cell fate is much more plastic than previously considered, and technologies for its manipulation are a keystone for many new tissue regeneration therapies. Transcription factors (TFs) are DNA-binding proteins that control gene expression, and they have critical roles in the control of cell fate and other cellular behavior. TF-based therapies have much medical potential, but their use as drugs depends on the development of suitable delivery technologies that can help them reach their action site inside of the cells. TFs can be used either as proteins or encoded in polynucleotides. When used in protein form, many TFs require to be associated to a cell-penetrating peptide or another transduction domain. As polynucleotides, they can be delivered either by viral carriers or by non-viral systems such as polyplexes and lipoplexes. TF-based therapies have extensively shown their potential to solve many tissue-engineering problems, including bone, cartilage and cardiac regeneration. Yet, their use has expanded beyond regenerative medicine to other prominent disease areas such as cancer therapy and immunomodulation. This review summarizes some of the delivery options for effective TF-based therapies and their current main applications.

Abstract 335: Distinct Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid Linked Phospholipids on Membrane Structure

In REDUCE-IT, prescription eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), significantly reduced ischemic events in patients with diabetes or cardiovascular disease. Treatments containing docosahexaenoic acid (DHA) have not shown similar benefits in other trials. EPA and DHA differentially influence lipid oxidation, signal transduction, fluidity, and cholesterol domain formation, potentially due to distinct membrane interactions. Small angle x-ray diffraction approaches compared the separate and combined effects of 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine (PL-EPA) and 1-palmitoyl-2-docosahexaenoic-sn-glycero-3-phosphocholine (PL-DHA) at a 1:20 ratio in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PL-OA) and cholesterol (C) at a 0.3:1 C:PL ratio. As a control, we also evaluated membranes consisting of PL-OA and C only. Electron density profiles (electrons/Å 3 vs Å) generated from the diffraction data served to determine membrane structure, including its width or d -space, at 1 Å resolution (Fig. 1A). Addition of PL-EPA increased membrane hydrocarbon core electron density over a broad area ± 0-10 Å from the membrane center, indicating an energetically favorable extended membrane orientation for EPA, stabilized by van der Waals interactions (Fig. 1B). By contrast, PL-DHA increased electron density in the phospholipid head group region concomitant with disordering in the hydrocarbon core ± 0-9 Å (Fig. 1C). Addition of the PL-O3FAs did not alter the overall membrane width (57 ± 1 Å). The combination of PL-EPA with PL-DHA highly attenuated their separate effects on the hydrocarbon core and headgroup (Fig. 1D). The contrasting EPA and DHA effects on membrane structure signify distinct molecular orientations that may contribute to observed differences in biological activity.

Recombinant Expression and Bioactivity Characterization of TAT-Fused Thymosin β10.

Thymosin beta 10 (TB10) is one of the common members among beta-thymosins. Human TB10 is reported to play a role in anti-angiogenesis and inhibition of cell migration during the tumorigenesis or metastasis of some certain cancers. Thus, it would be a potent clinical agent. In the present study, the coding sequence of TB10 was optimized based on the codon preference of Escherichia coli and cloned to pET28a (+) by chemical synthesis and molecular cloning methods. The recombinant protein was highly expressed employing E. coli expressing system and purified by a simple step of Ni2+ affinity chromatography. The TEV proteinase recognition site was inserted in the His6-tag and the target protein for easy removal of the His6-tag. To improve the biological activity of TB10, the transactivator of transcription (TAT) short peptide, a transduction domain, was added to the N-terminus of TB10. About 14.3mg of the recombinant TB10 proteins was obtained from 1L bacterial culture. The functional analyses demonstrated that the recombinant TB10 proteins displayed the distinct inhibition on angiogenesis by chick embryo chorioallantoic membrane assay and endothelial cell migration by wound healing assay. The TAT-fused TB10 even had stronger effects, probably due to the better transduction into the cells.

Abstract 159: Eicosapentaenoic Acid and Docosahexaenoic Acid Have Distinct Membrane Locations and Lipid Interactions as Determined by X-ray Diffraction

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) differentially influence lipid oxidation, signal transduction, fluidity, and cholesterol domain formation, potentially due to distinct membrane interactions. We used small angle x-ray diffraction to test EPA and DHA effects on model membrane structure. Vesicles were prepared to model human peripheral cell membranes using 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and cholesterol (C) (0.3 C:POPC mole ratio), and treated with vehicle, EPA, or DHA (1:10 mole ratio to POPC). Membrane width ( d- space) was 59.5 Å at 10°C, decreasing to 54.7 Å at 30°C due to increased acyl chain dynamics. EPA or DHA had no effect on membrane d -space (<1 Å change). Electron density profiles from diffraction data were superimposed on corresponding vehicle profiles (Fig. 1). EPA increased membrane hydrocarbon core electron density over a broad area, up to ± 20 Å from the membrane center, indicating an energetically favorable extended membrane orientation for EPA, stabilized by van der Waals interactions. By contrast, DHA increased electron density in the phospholipid head group region starting at ± 12 Å from the membrane center, due to DHA-surface interactions, resulting in a pronounced electron density reduction and increased disorder in the membrane hydrocarbon core centered ± 7-9 Å from the membrane center. DHA disordering effects were less apparent at higher temperatures, likely due to greater rotational dynamics, while EPA effects were stable. The contrasting EPA and DHA effects on membrane structure signify distinct molecular locations/orientations and may contribute to observed differences in biological activity.

Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult

BackgroundOxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1–6) that can reduce hydrogen peroxide (H2O2) and organic hydroperoxides to water and alcohols. MethodsWe produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis. ResultsOur data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H2O2-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H2O2-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage. ConclusionsThese findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo. General significancePEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.

Transient Alteration of Gene Expression in Adipose-Derived Stem Cells Using Liposomal-Driven Protein Extracts

Recent works have proposed the use of proteins instead of DNA/RNA as molecular tools for the modulation of gene expression patterns in stem cells. To obtain insulin-producing cells, certain proteins including Pdx-1, Isl-1, Pax-6, and Neuro-D, can be expressed or modified with transduction domains in order to penetrate and reprogram target cells. However, numerous aspects must be taken in account, including the fact that not all proteins may be used in transduction protocols. Thus, new methods must be developed. In this report we studied whether liposomes can work as carriers to introduce reprogramming protein extracts inside adipose tissue mesenchymal stem cells. To this end, the cells were incubated for 8 h in the presence of liposomes containing MIN-6 protein extracts. Charged liposomes fused with 70% of the cells, transferring their contents and transiently (up to 48 h) changing the gene expression pattern for specific endocrine pancreatic genes coding for Insulin-I, Insulin-II, Glucagon, Isl-1, MafA, and Nkx-6.1. No expression of Foxa-2, Pax-6, and Pdx-1 was observed. Although preliminary, the obtained results suggest that the use of liposomes for reprogramming techniques opens new possibilities for cell therapy.

Homeoproteins and Homeoprotein-derived Peptides: Going in and Out

Since the initial evidence that antennapedia homeobox can cross cell membranes and internalize into cells, numerous peptides with similar translocation properties have been described. These peptides are referred to as cell-penetrating peptides (CPPs) or protein-transduction domains (PTDs). Reviews on reported CPP sequences have been recently published, together with reviews on their mechanisms of internalization. In this review, we will focus on natural homeoproteins and homeoprotein-derived peptides and describe results that have been obtained among different laboratories to unravel the different pathways by which these molecules reach the cell cytosol and nucleus or transfer from one cell to another. Using homeoproteins as a paradigm, we will also summarize recent evidences of the physiological functions of endogenous protein translocation.

Cell-permeant peptide inhibitors of vasospasm and intimal hyperplasia

Outcomes from vein graft bypass are limited by graft failure, leading causes of which include intimal hyperplasia and vasospasm. Intimal hyperplasia remains the most common cause of graft failure, but no therapeutic modalities have been shown to prevent intimal hyperplasia in humans. The small heat shock proteins are a class of naturally occurring proteins in vascular smooth muscle. These proteins have an integral role in maintenance of vascular tone and in cellular defense against various stressors. Transduction domains have enabled intracellular therapeutic delivery of peptide analogs of heat shock proteins, as well as peptide inhibitors of the kinases that phosphorylate these proteins. These cell-permeant peptides have been shown to prevent vasospasm and intimal hyperplasia in vitro. Since vascular bypass using vein grafts is analogous to autologous organ transplantation, ex vivo treatment of the vein graft with cell-permeant peptide inhibitors of vasospasm and intimal hyperplasia prior to implantation provides a unique opportunity for targeted treatment of the graft to improve patency.

Abstract 36: Disrupting the Delta Protein Kinase C Interaction with the “d” Subunit of F1Fo ATP Synthase Protects Against Ischemia-Reperfusion Injury in Perfused Rat Hearts

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces >90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p<0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p<0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p < 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.

Transduction of Cell-Penetrating Peptides into Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells have recently been generated by Yamanaka's group, and then followed by others. iPS cells are expected to have clinical applications including an important role in regenerative medicine. This study focused on the cell-penetrating peptides (CPPs) for differentiation or functional application of iPS cells, because several transduction domains can deliver a large size-independent variety of molecules into cells. Two CPPs, Texas Red-R8 and Rhodamine-TAT, were generated as representative CPPs and these CPPs were tested to determine their ability to penetrate the membrane of iPS cells. Both CPPs were transduced in iPS cells through macropinocytosis classified in endocytosis within 2 h in a manner consistent with many other cells, and no cytotoxicity and influence on their undifferentiated state was observed. In conclusion, CPPs can be utilized for their differentiation or functional application in iPS cells.

P62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation.

Abstract 2857Poster Board II-833We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62−/− stromal cells with the full-length p62 construct restored the capacity of p62−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62−/− support of OCL formation. However, Δ2 construct transduced p62−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment. Disclosures:Roodman:Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.

Design of a bioactive cell‐penetrating peptide: when a transduction domain does more than transduce

The discovery of cell-penetrating peptides (CPPs) has facilitated delivery of peptides into cells to affect cellular behavior. Previously, we were successful at developing a phosphopeptide mimetic of the small heat shock-like protein HSP20 . Building on this success we developed a cell-permeant peptide inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2). It is well documented that inhibition of MK2 may be beneficial for a myriad of human diseases including those involving inflammation and fibrosis. During the optimization of the activity and specificity of the MK2 inhibitor (MK2i) we closely examined the effect of cell-penetrating peptide identity. Surprisingly, the identity of the CPP dictated kinase specificity and functional activity to an extent that rivaled that of the therapeutic peptide. The results reported herein have wide implications for delivering therapeutics with CPPs and indicate that judicious choice of CPP is crucial to the ultimate therapeutic success.

The Structure and Sequence Analysis of TLR4 Gene in Cattle

Toll-like receptor 4 (TLR4) is essential for initiating the innate response to lipopolysaccharide (LPS) from Gram-negative bacteria by acting as a signal transducting receptor. In order to help in investigating TLR4 as a candidate disease-resistance gene in cows, we isolated the cDNA (GenBank accession no. DQ839566) by RT-PCR and rapid amplification of cDNA ends (RACE) experiments and analyzed the sequence characters by bioinformatics. The results showed that cattle TLR4 gene about 3 739 bp contains an open reading frame of 2 526 bp encoded 841 amino acids (aa), 470 bp 5′ untranslated region (UTR), and 743 bp 3′ UTR. Tissue expression profile by RT-PCR indicated that TLR4 gene expresses in mammary glands, liver, muscle, duodenum, fats, uterus, kidneys, hearts, lungs, pancreas, and ovary. TLR4 protein domain predicted by bioinformatics consists of signal peptide, transmembrane helices domain, 3 sorts of leucine-rich repeat domains (LRR, LRR-TYP, and LRRCT), and a toll-interleukin1-resistance domain (TIR). Leucine-rich repeat domains were related with recognizing a broad of pathogen-associated molecular patterns (PAMP) from pathogen, and TIR domain for downstream signaling transduction was most conservative (98% identify) than other domains after alignment of protein from ovine, porcine, human, and mouse. In addition, a 470 bp 5′-flanking region sequence was amplified by PCR, and 15 putative DNA binding sites were predicted, but this sequence lacks TATA box, CCAAT character, and GC-rich regions.

Improving In Vivo Retention of Bioscavengers In Blood

We are developing technologies to increase the in vivo retention times of bioscavengers by attaching them to red blood cells (RBCs) using cell‐penetrating peptides (CPPs). CPPs are short peptides of 5‐40 amino acids, with the ability to translocate inside the cells by different mechanisms including direct physical transfer through the lipid bilayer. CPPs can either anchor the bioscavnegers to the cell surface, or internalize them by associated transport. Our results show that conjugation of the bioscavenger butyrylcholinesterase (BChE) to CPPs differently affected the enzymatic activity. Conjugation of Buforin I impaired the BChE activity by 50%. In contrast, the HIV Tat‐protein transduction domain (Tat) reduced the BChE activity by 11% in comparison to the unconjugated BChE. Tat was also found to significantly increase the association of BChE activity to RBC. Association of Tat to RBCs was also monitored by confocal microscopy using fluorescent microspheres. In addition, we are using artificial liposomes mimicking the RBC membrane composition as a further way to test CPP association to biological membranes. We are also investigating the capability of other RBC‐interacting peptides to associate bioscavengers. We expect that such technologies increase the effectiveness of protein therapeutics, or prophylactics, in blood borne treatments.Supported by DTRA#1.D0011_08_WR_C.

Relative expression of sarco‐endoplasmic reticulum calcium ATPase (SERCA) protein in crayfish, Procambarus clarkii during molting and in response to cold temperature

Molting is a prerequisite for development and growth in crustaceans and other arthropods. In the past we have used the natural molting cycle as a non‐mammalian model system to study calcium (Ca2+) homeostasis and the genes encoding for the Ca2+ handling proteins. Ca2+ pumps are integral membrane proteins having 1000 amino acid residues and three cytoplasmic domains (ATP‐binding site, phosphorylation site and a transduction domain). In both epithelial and non‐epithelial tissues of Procambarus clarkii, the Ca2+ pumping ATPase of sarcoplasmic reticulum (SERCA) performs the critical function of sequestering Ca2+ from the cytoplasm at the expense of ATP hydrolysis. In this study, we have specifically looked at the expression of mRNA and protein of SERCA in cardiac muscle during the molting cycle and cold acclimation at 4ºC for 3 weeks by using in‐situ hybridization and immunofluorescence. During intermolt and postmolt stages, a dynamic upregulation of SERCA expression was revealed in cardiac tissues as compared with premolt stage. Tissues of animals cold acclimated at 4ºC for 3 weeks also exhibited a significant increase in SERCA expression. These two experimental conditions will be further employed to better examine the mechanisms of regulation of expression of this important Ca2+ sequestration mechanism (Supported by Grant IBN 0445202 to MGW, YG and CMG).

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.

Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins.

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.