Kazuhito Tomizawa

Enhanced intracellular delivery using arginine-rich peptides by the addition of penetration accelerating sequences (Pas)

Cell penetrating peptides (CPPs), including arginine-rich peptides, are attractive tools for the intracellular delivery of various bioactive molecules with a low membrane permeability. We showed that the accelerated intracellular delivery of arginine-rich peptides was achieved by the addition of a short peptide segment (penetration accelerating sequence, Pas) to arginine-rich CPPs. The cytosolic release of the Pas-attached arginine-rich CPPs was observed within 5 min after the treatment of the cells with the peptides even in the presence of serum. Effectiveness of the Pas segment in the intracellular delivery of bioactive peptides using arginine-rich CPPs was exemplified through the enhanced growth inhibition activity of the malignant glioma cells by a retro-inverso peptide derived from the p53 C-terminal 22-amino-acid segment (positions 361-382).

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G0/G1 phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1T94E mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G1 length, cell cycle entry and fate determination of these cells during corticogenesis.

SIRT7 Controls Hepatic Lipid Metabolism by Regulating the Ubiquitin-Proteasome Pathway

Sirtuins (SIRT1-7) have attracted considerable attention as regulators of metabolism over the past decade. However, the physiological functions and molecular mechanisms of SIRT7 are poorly understood. Here we demonstrate that Sirt7 knockout mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance, and that hepatic triglyceride accumulation was also attenuated in liver-specific Sirt7 knockout mice. Hepatic SIRT7 positively regulated the protein level of TR4/TAK1, a nuclear receptor involved in lipid metabolism, and as a consequence activated TR4 target genes to increase fatty acid uptake and triglyceride synthesis/storage. Biochemical studies revealed that the DDB1-CUL4-associated factor 1 (DCAF1)/damage-specific DNA binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex interacted with TR4, leading to its degradation, while binding of SIRT7 to the DCAF1/DDB1/CUL4B complex inhibited the degradation of TR4. In conclusion, we propose that hepatic SIRT7 controls lipid metabolism in liver by regulating the ubiquitin-proteasome pathway.

Stabilization of Actin Bundles by a Dynamin 1/Cortactin Ring Complex Is Necessary for Growth Cone Filopodia

Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1–cortactin complexes appeared as “open” or “closed” rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1–cortactin complex.

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.Cysteine hydropersulfides (CysSSH) are believed to have a cellular redox protective role. Here the authors show that these species can be produced from L-cysteine by cysteinyl-tRNA synthetases and that these enzymes are also involved in mitochondrial biogenesis and bioenergetics regulation.

Oxytocin: A therapeutic target for mental disorders

We review here that oxytocin (OT) is released into blood and within distinct brain regions in response to stressful and social stimuli, and has been shown to have an antidepressant-like effect in animal studies. Clinical reports suggest OT to be a promising drug for psychiatric diseases such as depression, anxiety disorders, schizophrenia, and autism. OT may also have therapeutic potential in the treatment of major depressive disorders, even though OT administered into blood does not readily cross the blood–brain barrier. Physiological functions such as sexual activity and mating induce the release of OT in the central nervous system. A drug for the treatment of sexual dysfunction, sildenafil, enhances the electrically evoked release of OT from the posterior pituitary. This drug has antidepressant-like effects through activation of an OT signaling pathway. These results suggest that sildenafil may have promise as a potential antidepressant.

Cdk5rap1-Mediated 2-Methylthio Modification of Mitochondrial tRNAs Governs Protein Translation and Contributes to Myopathy in Mice and Humans

Transfer RNAs (tRNAs) contain a wide variety of posttranscriptional modifications that are important for accurate decoding. Mammalian mitochondrial tRNAs (mt-tRNAs) are modified by nuclear-encoded tRNA-modifying enzymes; however, the physiological roles of these modifications remain largely unknown. In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. The KO mice were highly susceptive to stress-induced mitochondrial remodeling and exhibited accelerated myopathy and cardiac dysfunction under stressed conditions. Furthermore, we demonstrate that the ms(2) modifications of mt-tRNAs were sensitive to oxidative stress and were reduced in patients with mitochondrial disease. These findings highlight the fundamental role of ms(2) modifications of mt-tRNAs in mitochondrial protein synthesis and their pathological consequences in mitochondrial disease.

Development of a bifunctional immunoliposome system for combined drug delivery and imaging in vivo.

The diverse characteristics of immunoliposomes provide advantages for utilization in drug delivery systems. In this study, we fused the antibody affinity motif of protein A (ZZ) with Gaussia luciferase (GLase). The fused protein conjugated with an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (GLase-ZZ-His-mAb) was effectively delivered into glioma cells expressing an activated EGFR mutant (EGFRvIII) and the bioluminescence was visualized in the cells. Immunoliposomes were further constructed with DSPE-PEG-MAL for covalent GLase-ZZ-His-mAb conjugation. A fluorescence dye (HPTS) encapsulated in immunoliposomes conjugated with GLase-ZZ-His-mAb was effectively delivered into EGFRvIII-expressing glioma cells. In a murine xenograft model of glioma, moreover, specific targeting of the immunoliposomes was visualized in the tumor. This new bifunctional immunoliposome system has the potential for drug delivery and imaging in vivo.

Identification of a splicing variant that regulates type 2 diabetes risk factor CDKAL1 level by a coding-independent mechanism in human

Single-nucleotide polymorphisms (SNPs) in CDKAL1 have been associated with the development of type 2 diabetes (T2D). CDKAL1 catalyzes 2-methylthio modification of adenosine at position 37 of tRNA(Lys)(UUU). A deficit of this modification causes aberrant protein synthesis, and is associated with impairment of insulin secretion in both mouse model and human. However, it is unknown whether the T2D-associated SNPs in CDKAL1 are associated with downregulation of CDKAL1 by regulating the gene expression. Here, we report a specific splicing variant of CDKAL1 termed CDKAL1-v1 that is markedly lower in individuals carrying risk SNPs of CDKAL1. Interestingly, CDKAL1-v1 is a non-coding transcript, which regulates the CDKAL1 level by competitive binding to a CDKAL1-targeting miRNA. By direct editing of the genome, we further show that the nucleotides around the SNP regions are critical for the alternative splicing of CDKAL1-v1. These findings reveal that the T2D-associated SNPs in CDKAL1 reduce CDKAL1-v1 levels by impairing splicing, which in turn increases miRNA-mediated suppression of CDKAL1. Our results suggest that CDKAL1-v1-mediated suppression of CDKAL1 might underlie the pathogenesis of T2D in individuals carrying the risk SNPs.

A protein transduction method using oligo-arginine (3R) for the delivery of transcription factors into cell nuclei

Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer.