Nahoko Kobayashi

Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

Efficient Generation of Dopamine Neuron-Like Cells From Skin-Derived Precursors With a Synthetic Peptide Derived From von Hippel-Lindau Protein

Skin-derived precursors (SKPs) from mammalian dermis represent neural crest-related stem cells capable of differentiating into both neural and mesodermal progency. SKPs are of clinical interest because they serve as accessible autologous donor cells for neuronal repair for neuronal intractable diseases. However, little is known about the efficient generation of neurons from SKPs, and phenotypes of neurons generated from SKPs have been restricted. In addition, the neuronal repair using their generated neurons as donor cells has not been achieved. The von Hippel-Lindau protein (pVHL) is one of the proteins that play an important role during neuronal differentiation, and recently neuronal differentiation of neural progenitor cells by intracellular delivery of a synthetic VHL peptide derived from elongin BC-binding site has been demonstrated. In the present study, a synthetic VHL peptide derived from elongin BC-binding site was conjugated to the protein transduction domain (PTD) of HIV-TAT protein (TATVHL peptide) to facilitate entry into cells, and we demonstrate the efficient generation of cells with dopaminergic phenotype from SKPs with the intracellular delivery of TATVHL peptide, and characterized the generated cells. The TATVHL peptide-treated SKPs expressed neuronal marker proteins, particularly dopamine neuron markers, and also up-regulated mRNA levels of proneural basic helix-loop-helix factors. After the TATVHL peptide treatment, transplanted SKPs into Parkinsons disease (PD) model rats sufficiently differentiated into dopamine neuron-like cells in PD model rats, and partially but significantly corrected behavior of PD model rats. The generated dopamine neuron-like cells are expected to serve as donor cells for neuronal repair for PD.

Nuclear Translocation Peptides as Antibiotics

Human immunodeficiency virus (HIV) Tat and antimicrobial peptides, such as LL-37, which transfer extraneous DNA to nucleus of host cells are known (5, 6). We performed experiments to find an antimicrobial function for nuclear localization signals (NLS) and nuclear export signals (NES), which are nuclear translocation signals required for shuttling cellular proteins between the nucleus and the cytoplasm (3, 4, 7). Consequently, we obtained evidence that the peptides, which consist of NLS tandems or combinations of NLS and NES, are effective antimicrobial peptides. The antimicrobial activity of these combination peptides for both gram-negative and gram-positive bacteria is very high, and antimicrobial peptides comprised of NLS and NES have the potential to become a new base for peptide antibiotics. The different combinations of NLS and NES within the peptidic design principle comprise eight types of results: the NLS simple sequence, the NLS tandem sequences (NLS+NLS and NLS+NLS+NLS), the NES simple sequence, the NES tandem sequence (NES+NES), the sequence composed of NES linked to an NLS C terminus (NLS+NES), the sequence composed of NLS linked to an NES C terminus (NES+NLS), and the sequence composed of NLS linked to an NLS+NES C terminus (NLS+NES+NLS). The eight types of peptides were synthesized by following the 9-fluorenylmethoxycarbonyl solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography on a C18 column (purity of ≥95%), and their antimicrobial function against the gram-positive bacterium Staphylococcus aureus IFO12732 and the gram-negative bacterium Escherichia coli IFO12713 was tested using the microbroth dilution method to evaluate their MICs. The results are shown in Table ​Table1.1. The antimicrobial activity for four types of NLS simple sequence (simian virus 40 [SV40] large T antigen, p54, SOX9, and NS5A) was present but weak. However, it was found that antimicrobial function increased remarkably when NLS was made into a tandem sequence (NLS+NLS). More specifically, the antimicrobial performance improved from 30 to 100 times that of NLS on its own. Moreover, the antimicrobial performance results against both gram-positive and gram-negative bacteria further improved several times by making NLS into the three NLS tandem sequence (NLS+NLS+NLS) and the NLS+NES+NLS tandem sequence. Also, NLS-linked NES (NLS+NES, NES+NLS) demonstrated a particularly strong antimicrobial effect against S. aureus. Compared to these results, the NES simple sequence and the NES tandem sequence (NES+NES) did not demonstrate a significant antimicrobial effect. TABLE 1. MICs for combination peptides of NLS and NES To evaluate the peptidic antimicrobial spectrum, MICs for five gram-positive bacterial strains and nine gram-negative bacterial strains were measured for the two types of NLS and NES combination peptides, and the results are shown in Table ​Table2.2. The highly efficient antimicrobial function of the peptide RKKKRKV RKKKRKV, the tandem sequence composed of the modified peptide from SV40 (Table ​(Table2)2) NLS (PKKKRKV), was also confirmed. It is a sequence pair where the Pro in the N terminus was replaced with Arg, showing that some rearrangement could produce better antimicrobial function in NLS/NES combination peptides. TABLE 2. MICs for combination peptides of NLS and NES for various gram-positive and gram-negative bacteria In this study, it was discovered that the antimicrobial function is inherent in the nuclear translocation signal peptide although this peptide does not originally exist for the purpose of self-defense. This is a significant difference from conventional antimicrobial peptides based on biomaterials that exist solely for self-defense (1, 2).

Nucleolar Localization Signals of LIM Kinase 2 Function as a Cell- Penetrating Peptide

LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLS-conjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that NoLS has potential for transporting biomolecules across the blood-brain barrier.

Engrafted VHL peptide-delivered bone marrow stromal cells promote spinal cord repair in rats

Stem cell-based therapy using bone marrow stromal cells (MSCs) has been expected to be a promising therapy for neuronal regeneration. To repair the injured spinal cord, neuronal differentiation of MSCs before transplantation has a more satisfactory effect. Recently, neuronal differentiation of neural progenitor/stem cells by an intracellular delivery of a pVHL-derived synthetic peptide (VHL peptide) has been shown. Here, we show that VHL peptide-delivered MSCs differentiated into neuron-like cells, and that engrafted VHL peptide-delivered MSCs more recovered the behaviors of the rats than that of nondelivered MSCs. Our result suggests that the use of VHL peptide-delivered MSCs would be a promising therapeutic strategy for repairing the injured spinal cord.

Binding Sites on Laminin Receptors as Components for Antibiotics

Bacteria use the receptor-adhesion-like interaction between laminin and the laminin receptor in the process of infection. We determined that bacteria do not interact with the receptor-binding site on laminin which could be expected for the bacterial laminin receptor. Rather, binding occurs via the laminin-binding site on the 67-kDa laminin receptor, which has a function similar to the one the bacterial laminin receptor possesses. This finding has implications for the effective use of antimicrobial peptides.

Efficient growth inhibition of human osteosarcoma cells using a peptide derived from the MDM-2-binding site of p53.

Protein transduction therapy is a promising alternative to gene therapy, but has not previously been investigated for osteosarcoma. We here demonstrate efficient growth inhibition of human osteosarcoma cells using a p53 peptide. Our result suggests that protein transduction therapy has significant potential as a novel therapeutic approach for osteosarcoma.

BC-Box Motif-Mediated Neuronal Differentiation of Somatic Stem Cells

Von Hippel-Lindau tumor suppressor protein (pVHL) functions to induce neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs). Here we identified a neuronal differentiation domain (NDD) in pVHL. Neuronal differentiation of SKPs was induced by intracellular delivery of a peptide composed of the amino-acid sequences encoded by the NDD. Neuronal differentiation mediated by the NDD was caused by the binding between it and elongin C followed by Janus kinase-2 (JAK2) ubiquitination of JAK2 and inhibition of the JAK2/the signal transducer and activator of transcription-3(STAT)3 pathway. The NDD in pVHL contained the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) corresponding to the binding site of elongin C. Therefore, we proposed that other BC-box proteins might also contain an NDD; and subsequently also identified in them an NDD containing the amino-acid sequence encoded by the BC-box motif in BC-box proteins. Furthermore, we showed that different NDD peptide-delivered cells differentiated into different kinds of neuron-like cells. That is, dopaminergic neuron-like cells, cholinergic neuron-like cells, GABAnergic neuron-like cells or rhodopsin-positive neuron-like cells were induced by different NDD peptides. These novel findings might contribute to the development of a new method for promoting neuronal differentiation and shed further light on the mechanism of neuronal differentiation of somatic stem cells.

Functional analyses of a novel vesicular membrane protein, TMEM141 during the growth stage in medaka (Oryzias latipes) larvae

genetic / environmental insults, potentially enhancing our understanding of the mechanism of developmental anomalies. Mathematical modelling has been used to determine base patterning programs operant in driving branching of the ureteric epithelium, facilitating the analysis of different genetic and morphological impacts on the development of the organ. Integrating cell, tissue and organ level datasets facilitates quantitative analysis of even subtle perturbations to kidney development and is also applicable to other organ systems.

Antiviral Properties of Combination Peptides of HIV-1 Rev NLS and NES

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Mareks disease virus serotype 1.