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Expression, purification, assay, and crystal structure of perdeuterated human arginase I.

ABSTRACT: Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to yield l-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) at 1.90A resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeuteration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study.

SUBMITTER: Di Costanzo L

PROVIDER: S-EPMC2018606 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Expression, purification, assay, and crystal structure of perdeuterated human arginase I.

Di Costanzo Luigi L Moulin Martine M Haertlein Michael M Meilleur Flora F Christianson David W DW

Archives of biochemistry and biophysics 20070521 1

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to yield l-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) ...[more]

PMID: 17562323

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Project description:BackgroundProstate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.Methodology/principal findingsIn this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.Conclusion/significanceOur data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.

Dataset Information

Expression, purification, assay, and crystal structure of perdeuterated human arginase I.

Publications

Expression, purification, assay, and crystal structure of perdeuterated human arginase I.

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