Mammalian transglutaminase (TGase) catalyzes covalent crosslinking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPD(P), QxP, and QxxDP; Factor XIIIa, QxxxWP (where x and represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.Transglutaminase (TGase, 2 2.3.2.13) is an enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and also attaches other primary amines to peptide-bound glutamines (1-5). To date, eight human TGase isozymes (Factor XIII, TGases 1-7) have been identified, comprising a large protein family with unique tissue distributions and physiological roles. Among the isozymes, TGase 2 and Factor XIII are two major members, although their locations and regulation differ. TGase 2 is expressed ubiquitously and has been implicated in many biological processes, including apoptosis, stabilization of the extracellular matrix, and regulation of growth factors (6, 7). Plasma Factor XIII is synthesized as a zymogen that comprises two A and two B subunits (8). The A subunit contains the catalytic domain and is converted by thrombin-dependent proteolysis into its active form during clot formation. Activated Factor XIII (Factor XIIIa) is involved in fibrin stabilization and wound healing (9).Generally, TGase reactions involve a Ca 2ϩ -dependent acyl transfer via a double displacement mechanism. In the first step, a glutamine ␥-carboxyamide group in the substrate binds to a cysteine at the active site, resulting in formation of a ␥-glutamylthioester bond and release of ammonia. Formation of the covalent acyl enzyme intermediate is the rate-limiting step. The -amino group of a peptide-bound lysine, as a nucleophilic substrate, binds to the acyl enzyme intermediate and then attacks the thioester bond, thereby generating an intermolecular isopeptide -(␥-glutamyl)lysine cross-link. Primary amines can replace lysine in transamidation reactions and become incorp...

CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate‐cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9‐expressing cancer cells. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS‐mediated apoptosis. Twenty non‐pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK‐1A and HAK‐1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC‐treated tissues (5/7) than in non‐pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9‐harboring HAK‐1B cells through ROS‐mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS‐mediated apoptosis in CDDP‐treated HCC cells, in which the CD44v9‐xCT system functioned. As CD44v9 is typically expressed in HAIC‐resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance.

Doublecortin-like kinase 1 (DCLK1), a marker for intestinal and pancreatic cancer stem cells, is highly expressed in neuroblastomas. This study was conducted to assess DCLK1 expression levels in pancreatic neuroendocrine tumor (PNET) tissues and to explore the roles of this molecule in clinical tissue from multiple PNET patients, cells (BON1, QGP1, and CM) and tumor xenografts. Immunohistochemically, all PNET tissues highly and diffusely expressed DCLK1 as a full-length isoform, identical to that detected in primary liver NETs. A DCLK1-overexpressing PNET cell line (QGP1-DCLK1) exhibited epithelial-mesenchymal transition (EMT)-related gene signatures, and robust upregulation of Slug (SNAI2), N-Cadherin (CDH2), and Vimentin (VIM) was validated by real-time PCR and immunoblotting. QGP1-DCLK1 cells had increased cell migration in a wound-healing assay and formed significantly larger xenograft tumors in nude mice. The factors involved in the formation of the fast-growing tumors included p-FAK (on Tyr925), p-ERK1/2, p-AKT, Paxillin, and Cyclin D1, which upon knockdown or pharmacologic inhibition of DCLK1 abolished the expression of these molecules. In conclusion, robust and ubiquitous expression of DCLK1 was first demonstrated here in human PNET tissue specimens and cells. DCLK1 characterized the PNET cell behavior, inducing p-FAK/SLUG-mediated EMT. These findings suggest the possibility of developing novel therapeutic strategies against PNETs by targeting DCLK1. Evidence here reveals that human PNETs diffusely and robustly express the cancer stem cell marker DCLK1, which drives SLUG-mediated EMT, and suggests that NETs share biological features for druggable targets with other tumors, including neuroblastoma that also highly expresses DCLK1. .

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During axon guidance, growth cones navigate toward attractive cues by inserting new membrane on the cue side. This process depends on Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) channels, but the Ca(2+) sensor and effector governing this asymmetric vesicle export remain unknown. We identified a protein complex that controls asymmetric ER Ca(2+)-dependent membrane vesicle export. The Ca(2+)-dependent motor protein myosin Va (MyoVa) tethers membrane vesicles to the ER via a common binding site on the two major ER Ca(2+) channels, inositol 1,4,5-trisphosphate and ryanodine receptors. In response to attractive cues, micromolar Ca(2+) from ER channels triggers MyoVa-channel dissociation and the movement of freed vesicles to the cue side, enabling growth cone turning. MyoVa-Ca(2+) channel interactions are required for proper long-range axon growth in developing spinal cord in vivo. These findings reveal a peri-ER membrane export pathway for Ca(2+)-dependent attraction in axon guidance.