Geetha M. Habib

Ubiquitination of Keap1, a BTB-Kelch Substrate Adaptor Protein for Cul3, Targets Keap1 for Degradation by a Proteasome-independent Pathway

Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.

Disruption of γ-Glutamyl Leukotrienase Results in Disruption of Leukotriene D4 Synthesis In Vivo and Attenuation of the Acute Inflammatory Response

ABSTRACT To study the function of γ-glutamyl leukotrienase (GGL), a newly identified member of the γ-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGLtm1) and in both GGL and GGT (GGLtm1-GGTtm1) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGLtm1 show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but ∼70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C4 (LTC4) to LTD4, indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD4. Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC4 and LTD4 have distinctly different functions in the inflammatory process.

γ-Glutamyl Leukotrienase, a Novel Endothelial Membrane Protein, Is Specifically Responsible for Leukotriene D4 Formation in Vivo

The metabolism of cysteinyl leukotrienes in vivo and the pathophysiological effects of individual cysteinyl leukotrienes are primarily unknown. Recently we identified an additional member of the γ-glutamyl transpeptidase (GGT) family, γ-glutamyl leukotrienase (GGL), and developed mice deficient in this enzyme. Here we show that in vivo GGL, and not GGT as previously believed, is primarily responsible for conversion of leukotriene C4 to leukotriene D4, the most potent of the cysteinyl leukotrienes and the immediate precursor of leukotriene E4. GGL is a glycoprotein consisting of two polypeptide chains encoded by one gene and is attached at the amino terminus of the heavy chain to endothelial cell membranes. In mice it localizes to capillaries and sinusoids in most organs and in lung to larger vessels as well. In contrast to wild-type and GGT-deficient mice, GGL-deficient mice do not form leukotriene D4 in vivo either in blood when exogenous leukotriene C4 is administered intravenously or in bronchoalveolar lavage fluid of Aspergillus fumigatus extract-induced experimental asthma. Further, GGL-deficient mice show leukotriene C4 accumulation and significantly more airway hyperreponsiveness than wild-type mice in the experimental asthma, and induction of asthma results in increased GGL protein levels and enzymatic activity. Thus GGL plays an important role in leukotriene D4 synthesis in vivo and in inflammatory processes.

Identification of two additional members of the membrane-bound dipeptidase family

We have cloned two mouse cDNAs encoding previously unidentified membrane‐bound dipeptidases [membrane‐bound dipeptidase‐2 (MBD‐2) and membrane‐bound dipeptidase‐3 (MBD‐3)] from membrane‐bound dipeptidase‐1 (MBD‐1) deficient mice (Habib, G.M., Shi, Z‐Z., Cuevas, A.A., Guo, Q., Matzuk, M.M., and Lieberman, M.W. (1998) Proc. Natl. Acad. Sci. USA 95, 4859–4863). These enzymes are closely related to MBD‐1 (EC 3.4.13.19), which is known to cleave leukotriene D4 (LTD4) and cystinyl‐bis‐glycine. MBD‐2 cDNA is 56% identical to MBD‐1 with a predicted amino acid identity of 33%. The MBD‐3 and MBD‐1 cDNAs share a 55% nucleotide identity and a 39% predicted amino acid sequence identity. All three genes are tightly linked on the same chromosome. Expression of MBD‐2 and MBD‐3 in Cos cells indicated that both are membrane‐bound through a glycosylphosphatidyl‐inositol linkage. MBD‐2 cleaves leukotriene D4 (LTD4) but not cystinyl‐bis‐glycine, while MBD‐3 cleaves cystinyl‐bis‐glycine but not LTD4. MBD‐1 is expressed at highest levels in kidney, lung, and heart and is absent in spleen, while MBD‐2 is expressed at highest levels in lung, heart, and testis and at somewhat lower levels in spleen. Of the tissues examined, MBD‐3 expression was detected only in testis. Our identification of a second enzyme capable of cleaving LTD4 raises the possibility that clearance of LTD4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.

Four Distinct Membrane-bound Dipeptidase RNAs Are Differentially Expressed and Show Discordant Regulation with γ-Glutamyl Transpeptidase

Membrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a γ-glutamyl group by γ-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5′ flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes.

Cloning of cDNA and genomic structure of the mouse γ-glutamyl transpeptidase-encoding gene

Abstract We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse γ-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exons and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.

Mouse leukotriene A4 hydrolase is expressed at high levels in intestinal crypt cells and splenic lymphocytes

LTA4 hydrolase (EC 3.3.2.6) is a dual-function enzyme that is essential for the conversion of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) and also possesses an aminopeptidase activity. To characterize the expression of this unusual enzyme, we have cloned the mouse LTA4 hydrolase cDNA. The deduced amino acid sequence revealed 92% identity with the human sequence. Cloning and analysis of genomic sequences of mouse LTA4 hydrolase indicated that it is a single-copy gene spanning over 40kb and containing 20 exons. LTA4 hydrolase is widely expressed, with the highest levels of expression occurring in the small intestine, followed by the spleen. In situ hybridization revealed that LTA4 hydrolase is localized in the crypt cells of the small intestine, white pulp of the spleen, bronchiolar epithelium of the lung, myocardium, adrenal cortex, epithelium of the seminal vesicles, proximal tubules and the collecting ducts of the kidney, and occasional hepatocytes. Thus the widespread distribution of LTA4 hydrolase in various cell types in the tissues suggests that LTB4 may possess biological activities other than those known at present. It is also plausible that the widespread occurrence of LTA4 hydrolase in various tissues may correspond more with its function as an aminopeptidase than its function as an LTA4 hydrolase.

Cleavage of Leukotriene D4 in Mice with Targeted Disruption of a Membrane-Bound Dipeptidase Gene

Leukotriene C4 (LTC4), leukotriene D4 (LTD4), and leukotriene E4 (LTE4), collectively known as cysteinyl leukotrienes, are members of the eicosanoid group of lipid mediators1. They have been implicated in a wide variety of acute and chronic inflammatory conditions including asthma, tissue injury, cardiac and liver diseases, and shock2,3. Production of LTC4 is initiated by the conjugation of the epoxide intermediate LTA4 with glutathione (GSH). LTC4 is further metabolized to the cysteinyl glycine conjugate of LTA4 known as LTD4 by the actions of two plasma-membrane-bound ectoenzymes, γ-glutamyl transpeptidase (GGT) and γ-glutamyl leukotrienase (GGL)4,5. LTD4 is believed to be converted to the less active cysteinyl glycine conjugate of LTA4 called LTE4 by a dipeptidase6. The rank order of molar potencies of cysteinyl leukotrienes is LTD4>LTC4 >LTE4. LTD4 is considered to be at least 10 to 100-fold more potent than LTE4 7. Consequently, conversion of LTD4 to LTE4 is a critical step in the cysteinyl leukotriene metabolism. LTC4, LTD4, and LTE4 are eliminated from the blood circulation with initial half-lives of 30–40 s. They are mainly taken up by kidney and liver and excreted into the urine and bile, respectively8. Thus, the liver seems to be the major site of their metabolic inactivation.