Isothiocyanate groups of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibit cell penetration of octa-arginine (R8)-fused peptides.

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine-a well-known cell-penetrating peptide (CPP)-is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide-ie, mitochondrial targeting domain (MTD)-to the cytosol. We found that 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonate (DIDS)-an anion channel blocker-inhibited the ability of octa-arginine (R8)-fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di-sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di-sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.

A peptide containing Noxa mitochondrial-targeting domain induces cell death via mitochondrial and endoplasmic reticulum disruption.

Noxa is a weak apoptosis activator consisting of a BH3 domain and a mitochondrial-targeting domain (MTD). BH3 binds Mcl-1 and Bcl2A1 and inactivates their anti-apoptotic activities, while MTD delivers BH3 to mitochondria. Previously we revealed that MTD may also function as an inducer of necrosis via conjugation with octa-arginine, which induces cytosolic Ca2+ influx from mitochondria. However, the mechanism(s) underlying this process has not been elucidated yet. Here, we show that calcium influx induced by an MTD peptide fused with octa-arginine residue (R8:MTD) originates not only from mitochondria but also from the extracellular space. However, calcium spikes were not sufficient for necrosis. R8:MTD induced mitochondrial permeability transition pore opening, fragmentation, and swelling. These mitochondrial events induced by MTD appeared to be necessary for necrosis induction, since DIDS, a VDAC inhibitor, inhibited the mitochondrial swelling and cell death induced by MTD. We show that R8:MTD disrupted endoplasmic reticulum (ER) structures but not peroxisomes or Golgi, indicating that R8:MTD causes necrosis by inducing ER events as well.

MTD-like motif of a BH3-only protein, BNIP1, induces necrosis accompanied by an intracellular calcium spike.

The mitochondrial targeting domain (MTD) of Noxa has necrosis-inducing activity when conjugated with cell-penetrating peptide (CPP). In this study, we report another MTD-like motif, B1MLM, found in BNIP1, a pro-apoptotic BH3-only protein found in the endoplasmic reticulum membrane. The B1MLM peptide, conjugated with CPP, induced necrosis in a way similar to that of R8:MTD. R8:B1MLM caused an intracellular calcium spike, mitochondrial reactive oxygen species generation, and mitochondrial fragmentation. The cytosolic calcium spike was likely due to the opening of the mitochondrial permeability transition pore.

Suppression of neointimal hyperplasia induced by arteriovenous anastomosis and balloon injury in rats by multimeric tumor necrosis factor-related apoptosis-inducing ligand.

Excessive blood vessel wall thickening, known as intimal hyperplasia, can result from injury or inflammation and increase the risk of vascular diseases. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays key roles in tumor surveillance, autoimmune diseases, and apoptosis; however, its role in vascular stenosis remains controversial. Treatment with recombinant isoleucine zipper hexamerization domain soluble TRAIL (ILz(6):TRAIL) significantly inhibited the progression of neointimal hyperplasia (NH) induced by anastomosis of the carotid artery and jugular vein dose dependently, and adenovirus expressing secretable ILz(6):TRAIL also inhibited NH induced by balloon injury in the femoral artery of rats. This study demonstrated the preventive and partial regressive effects of ILz(6):TRAIL on anastomosis of the carotid artery and jugular vein- or balloon-induced NH.

Rapid production method with increased yield of high-purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide.

Extracellular vesicles (EVs) are nano-sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large-scale production of high-purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30-fold) in EV production with markedly high purity (approximately 45-fold). These EVs (TS-eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA-AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS-eEV production induced by eMTDΔ4, indicating that the eMTDΔ4-mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS-eEVs. The present study may lead to considerable advances in EV-based drug development and production of stem cell-derived EVs for cell therapy.

Mitochondrial Targeting Domain Homologs Induce Necrotic Cell Death Via Mitochondrial and Endoplasmic Reticulum Disruption.

The mitochondrial targeting domain (MTD) of Noxa contributes to its mitochondrial localization and to apoptosis induction. As a peptide, MTD fused with octa-arginine (R8), a CPP, induces necrosis related to intracellular calcium influx and destruction of mitochondria and endoplasmic reticulum. We searched for homologs of MTD, and compared their cell killing capability when fused with R8. Three of the seven peptides triggered cell death with similar mechanisms. The comparative analysis of peptide sequences showed that four amino acid sites of MTD are critical in regulating necrosis, suggesting the potential to generate artificial, adjustable cytotoxic peptides, which could be effective medicines for many diseases. Thus, homologs functionality could hint to the functions of their belonging proteins.

Development of the Mammalian Expression Vector System that can be Induced by IPTG and/or Lactose.

Techniques used for the regulation of gene expression facilitate studies of gene function and treatment of diseases via gene therapy. Many tools have been developed for the regulation of gene expression in mammalian cells. The Lac operon system induced with isopropyl ß-D-1- thiogalactopyranoside (IPTG) is one of the employed inducible systems. IPTG mimics the molecular structure of allolactose and has a strong affinity for the corresponding repressor. IPTG is known to rapidly penetrate into mammalian cells and exhibits low toxicity. In the present study, we developed a new inducible expression system that could regulate the expression of genes in mammalian cells using IPTG. Here we confirm that unlike other vector systems based on the Lac operon, this expression system allows regulation of gene expression with lactose in the mammalian cells upon transfection. The co-treatment with IPTG and lactose could improve the regulatory efficiency of the specific target gene expression. The regulation of gene expression with lactose has several benefits. Lactose is safe in humans as compared to other chemical substances and is easily available, making this technique very cost-effective.

Noxa mitochondrial targeting domain induces necrosis via VDAC2 and mitochondrial catastrophe.

Noxa, a Bcl-2 homology 3 (BH3)-only protein of the Bcl-2 family, is responsive to cell stresses and triggers apoptosis by binding the prosurvival Bcl-2-like proteins Mcl1, BclXL, and Bcl2A1. Although the Noxa BH3 domain is necessary to induce apoptosis, the mitochondrial targeting domain (MTD) of Noxa functions as a pronecrotic domain, an inducer of mitochondrial fragmentation, and delivery to mitochondria. In this study, we demonstrate that the extended MTD (eMTD) peptide induces necrotic cell death by interaction with the VDAC2 protein. The eMTD peptide penetrates the cell membrane, causing cell membrane blebbing, cytosolic calcium influx, and mitochondrial swelling, fragmentation, and ROS generation. The MTD domain binds VDACs and opens the mitochondrial permeability transition pore (mPTP) in a CypD-independent manner. The opening of mPTP induced by eMTD is inhibited either by down-regulation of VDAC2 or by the VDACs inhibitor DIDS. These results indicate that the MTD domain of Noxa causes mitochondrial damage by opening mPTP through VDACs, especially VDAC2, during necrotic cell death.