Joohong Park

Stromal cell-derived factors 1α and 1β, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8

N‐terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G‐protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N‐terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF‐1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.

The In Vivo Expression of Dipeptidyl Peptidases 8 and 9

The dipeptidyl peptidase IV (DPIV) enzyme family contains both potential and proven therapeutic targets. Recent reports indicate the presence of DP8 and DP9 in peripheral blood lymphocytes, testis, lung, and brain. For a more comprehensive understanding of DP8 and DP9 tissue and cellular expression, mRNA and enzyme activity were examined. Many organs from C57BL/6 wild-type and DPIV gene-knockout mice were examined; DP8/9 enzyme activity was detected in the immune system, brain, testis, muscle, and epithelia. In situ hybridization localized DP8 and DP9 mRNA to lymphocytes and epithelial cells in liver, gastrointestinal tract, lymph node, spleen, and lung. DP8 and DP9 mRNA was detected in baboon and mouse testis, and DP9 expression was elevated in human testicular cancers. DP8 and DP9 mRNA were ubiquitous in day 17 mouse embryo, with greatest expression in epithelium (skin and gastrointestinal tract) and brain. Thus, DP8 and DP9 are widely expressed enzymes. Their expression in lymphocytes and epithelia indicates potential for roles in the digestive and immune systems. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Oral nicotinamide protects against ultraviolet radiation-induced immunosuppression in humans

Cutaneous immunity, which is a key defence against the development of skin cancers, is suppressed by even small doses of ultraviolet (UV) radiation. Preventing this UV-induced immunosuppression may therefore reduce the incidence of skin cancer. Nicotinamide (vitamin B3) has immune-protective and cancer-preventive effects against UV radiation in mice, and we have shown previously that topical nicotinamide is immune protective in humans. Using the Mantoux model of skin immunity in healthy volunteers, we compared oral nicotinamide to placebo (both administered for 1 week) in a randomized, double-blinded, crossover design against the effects of solar-simulated ultraviolet (ssUV) radiation on delayed-type hypersensitivity to tuberculin purified protein derivative. Discrete areas of the back were irradiated with low doses of ssUV daily for three consecutive days. Immunosuppression, calculated as the difference in Mantoux-induced erythema of irradiated sites compared with unirradiated control sites, was determined in volunteers taking oral nicotinamide and placebo. Significant immunosuppression occurred in an UV dose-dependent manner in the presence of placebo. Oral nicotinamide, at doses of either 1500 or 500 mg daily, was well tolerated and significantly reduced UV immunosuppression with no immune effects in unirradiated skin. Oral nicotinamide is safe and inexpensive and looks promising as a chemopreventive supplement for reducing the immunosuppressive effects of sunlight.

Topical nicotinamide modulates cellular energy metabolism and provides broad-spectrum protection against ultraviolet radiation-induced immunosuppression in humans

Background  Ultraviolet (UV) radiation can profoundly suppress the cutaneous immune system, thus enhancing carcinogenesis. Agents that prevent UV‐induced immunosuppression may thus reduce skin cancer. Nicotinamide (vitamin B3) prevents UV‐induced immunosuppression and carcinogenesis in mice, and solar‐simulated (ss) UV‐induced immunosuppression in humans. Its effectiveness against different UV wavebands and mechanism of action is as yet unknown.

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV‐induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism‐related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV‐induced cellular ATP loss and protected against UV‐induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV‐induced oxidative stress posttranslationally, we also measured UV‐induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

Hydrogen peroxide (H2O2) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H2O2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H2O2, N-ethylmaleimide or pyrroloquinoline qui- none. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism in- volves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H2O2 sensing proteins.

Structure and Function in Dipeptidyl Peptidase IV and Related Proteins

Potential therapeutic applications of DPIV inhibitors have fuelled interest in understanding the biological roles of DPIV and its relatives. Such efforts are confounded by the ubiquitous expression of DPIV, inhibitor selectivity questions and the variety of identified substrates. DPIV is not essential, but is such a useful enzyme that all animal species express it. The enzyme activity’s ancient and primary function is probably nutritional, providing more complete proteolysis of food and recycled proteins. This function is unnecessary in well-fed humans. The development of selective inhibitors of proteolytic activity and identification of ligand binding activities in this gene family would lead to rapid advances in understanding the biology of the POP gene family.

Outcomes after endovascular treatment of symptomatic patients with Takayasu's arteritis.

We report our experience with endovascular treatment of supra-aortic arteries and follow-up results in patients with Takayasus arteritis (TA) presenting with neurological symptoms. Of the 20 patients with TA who underwent cerebral angiography for neurological manifestations between May 2002 and May 2009, 12 (11 females, one male; mean age, 39 years; range 31–56 years) underwent endovascular treatment and evaluated outcome for 21 lesions, including nine common carotid arteries, four vertebral arteries, four subclavian arteries, two internal carotid arteries, and one brachiocephalic artery. Eight patients underwent multiple endovascular procedures for different lesions in single or multiple stages. Mean angiographic and clinical follow-up durations were 34 months (range, 11–79 months) and 39 months (range 11–91 months), respectively. Technical success was achieved for 20 procedures in 11 patients. One procedure failed, with 50% residual stenosis after stenting due to dense calcification of vessel walls. There were no procedure-related complications. Restenosis occurred at two lesions in two patients were treated by re-stenting. Asymptomatic occlusion occurred at two lesions in one patient. Ten patients remained in 0–1 on the modified Rankin scale (mRs) during mean 39 months. One patient, however, had a score of 3 on mRs due to a traumatic contusion during follow-up. One patient died from cardiac failure 36 months after successful angioplasty. Our data suggest that endovascular treatment of symptomatic supra-aortic lesions of TA is effective and durable in selected patients with neurologic symptoms.

Design and synthesis of novel inhibitors of human kynurenine aminotransferase-I

Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 μM.

High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate

In this study, we report two high‐resolution structures of the pyridoxal 5′ phosphate (PLP)‐dependent enzyme kynurenine aminotransferase‐I (KAT‐I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT‐I at 1.28 Å resolution, and the other with the general PLP‐dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP‐bound structures. We also report the inhibition of KAT‐1 by AOAA and aminooxy‐phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT‐I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.