Inhibition of Major Histocompatibility Complex Class I Antigen Presentation in Pig and Primate Cells by Herpes Simplex Virus Type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8⁺ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8⁺ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species—mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans—and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8⁺-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8⁺ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47⁻ mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.Herpes simplex virus (HSV) infection of human fibroblasts leads to inhibition of antigen presentation to CD8⁺ T cells so that the virus-infected fibroblasts are not lysed by cytotoxic T lymphocytes (CTL) (10, 12, 14). The principal reason for this resistance to CTL appears to be the expression of an HSV immediate-early protein, ICP47, which causes major histocompatibility complex (MHC) class I proteins to accumulate in infected cells in a peptide-empty form (19). ICP47 was subsequently shown to inhibit the transporter associated with antigen presentation (TAP), which functions to translocate antigenic peptides across the membrane of the endoplasmic reticulum (ER) (3, 8), and without antigenic peptides, MHC class I proteins accumulate in the ER. More recent results demonstrated that ICP47 blocks peptide binding to TAP by binding with high affinity to a domain of TAP that includes the peptide binding site (1, 15).Although HSV type 1 (HSV-1) ICP47 (ICP47-1) effectively blocks TAP in human fibroblasts, it inhibits TAP little, if at all, in a variety of mouse cells unless applied in high concentrations (1, 3, 15, 19). Similarly, HSV-2 ICP47 (ICP47-2), which has only 42% amino acid identity with ICP47-1 (4), effectively blocks human TAP but inhibits murine TAP less effectively (16). Inhibition of murine TAP with these proteins occurs at ICP47-1 and ICP47-2 concentrations 50- to 100-fold higher than those required to inhibit human TAP. ICP47-1 and ICP47-2 bind poorly to mouse TAP (15, 16), which explains their inability to block peptide transport and antigen presentation in mouse cells.We were interested in extending the study of the species specificity of ICP47 for several reasons. Firstly, we wanted to find an animal model with which to assess the effects of ICP47 in vivo, both to assess its role in virus-host interactions and to provide a model for the use of ICP47 in autoimmunity, in transplantation, and in gene therapy vectors. Secondly, we wanted to determine whether ICP47 was functional in the species currently widely used for HSV pathogenesis and vaccine studies—mice, rabbits, and guinea pigs. Thirdly, we were interested in the mechanism of the extraordinary virulence of HSV in owl monkeys (aotus), speculating that the TAP in this New World primate might be exceptionally susceptible to ICP47.In order to assess the effects of ICP47 on the TAPs of various species, cells were permeabilized, recombinant ICP47-1 and ICP47-2 were introduced into the cells, and assays of TAP activity were performed. To examine the effects of ICP47 on antigen presentation and recognition by CD8⁺ T cells, fibroblasts were infected with recombinant HSV-1 that expresses mouse class I proteins and not ICP47, and lysis of the cells by mouse anti-HSV CTL was tested. We found that ICP47-1 and ICP47-2 did not block TAP in mouse, rat, guinea pig, or rabbit skin fibroblasts but effectively inhibited TAP and antigen presentation in pig, dog, cow, and monkey fibroblasts. Therefore, pigs, dogs, and monkeys can be used to study the in vivo effects of ICP47, though for several reasons, the use of pigs might be a practical starting point.     

Resistance of Human Cytomegalovirus to Benzimidazole Ribonucleosides Maps to Two Open Reading Frames: UL89 and UL56

2,5,6-Trichloro-1-β-d-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused ∼10-fold resistance to the benzimidazoles and that the combination of both mutations caused ∼30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259–2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.Human cytomegalovirus (HCMV) can cause significant morbidity and mortality in immunocompromised populations (3). It is a common opportunistic disease in patients with AIDS and is often a factor in their death (38). HCMV infection has been implicated in increased risk of organ rejection following heart (28) and kidney transplants (8) and in restenosis of diseased arteries following angioplasty (41, 63). It is also a leading cause of birth defects (16).Current therapies for HCMV infection include ganciclovir (GCV) (22), cidofovir (30), and foscarnet (20). Each of these drugs has several limitations to its use: none are orally bioavailable, all have dose-limiting toxicity, and resistance has developed to each (26). Because all three of these drugs inhibit viral replication through an interaction with the virally encoded DNA polymerase (25, 31, 37), the possibility of cross-resistance exists. Thus, additional drugs with unique mechanisms of action are needed for the treatment of HCMV infections.In 1995, we reported that 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB; Fig. ​Fig.1)1) and the 2-chloro analog [2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole TCRB] are potent and selective inhibitors of HCMV replication (55). These compounds have a novel mechanism of action, which unlike the current therapies for HCMV infection, does not involve inhibition of DNA synthesis. The benzimidazole ribonucleosides prevent the cleavage of high-molecular-weight viral DNA concatemers to monomeric genomic lengths (57). Resistance to BDCRB has been mapped to the HCMV UL89 open reading frame (ORF), which, by analogy to gene gp17 from bacteriophage T4, may be a terminase (23, 57). Consequently, we have proposed that the benzimidazole ribonucleosides inhibit the product of this gene and that the UL89 gene product is involved in the viral DNA concatemer cleavage process (57). Open in a separate windowFIG. 1Structure of benzimidazole ribonucleosides. TCRB, R = Cl; BDCRB, R = Br.HCMV replication proceeds in a manner which is conserved among herpesviruses. The virally encoded DNA polymerase produces large, complex head-to-tail concatemers (10, 29, 33) which must be cleaved into genomic-length pieces before insertion into preformed capsids (59). With herpes simplex virus type 1 (HSV-1), temperature-sensitive mutants which are unable to cleave and package the concatemeric DNA have been derived (1, 2, 4, 45, 49, 50, 61). By this process, six HSV-1 genes have been found to be involved in concatemer cleavage and packaging. They are UL6, UL15, UL25, UL28, UL32, and UL33. In addition, recent studies in Homa’s laboratory have established that the product of UL25 is required for viral DNA encapsidation but not cleavage (39). Homologs of these genes exist in HCMV and are UL104, UL89, UL77, UL56, UL52, and UL51, respectively (18).In our continuing investigation of the mode of action of benzimidazole nucleosides, we report herein the independent isolation of HCMV strains resistant to TCRB, characterization of these strains, and identification of the mutations responsible for the development of resistance. The results demonstrate that the mechanism of action of the benzimidazole ribonucleosides is more complex than previously proposed and that a second gene product implicated in DNA cleavage and packaging is involved.     

Mutational Biosynthesis of Novel Rapamycins by a Strain of Streptomyces hygroscopicus NRRL 5491 Disrupted in rapL,Encoding a Putative Lysine Cyclodeaminase

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added l-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific l-lysine cyclodeaminase important for the production of the l-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.Rapamycin is a 31-member macrocyclic polyketide produced by Streptomyces hygroscopicus NRRL 5491 which, like the structurally related compounds FK506 and immunomycin (Fig. ​(Fig.1),1), has potent immunosuppressive properties (24). Such compounds are potentially valuable in the treatment of autoimmune diseases and in preventing the rejection of transplanted tissues (16). The biosynthesis of rapamycin requires a modular polyketide synthase, which uses a shikimate-derived starter unit (11, 20) and which carries out a total of fourteen successive cycles of polyketide chain elongation that resemble the steps in fatty acid biosynthesis (2, 27). l-Pipecolic acid is then incorporated (21) into the chain, followed by closure of the macrocyclic ring, and both these steps are believed to be catalyzed by a pipecolate-incorporating enzyme (PIE) (18), the product of the rapP gene (8, 15). Further site-specific oxidations and O-methylation steps (15) are then required to produce rapamycin. Open in a separate windowFIG. 1Structures of rapamycin, FK506, and immunomycin.The origin of the pipecolic acid inserted into rapamycin has been previously established (21) to be free l-pipecolic acid derived from l-lysine (although the possible role of d-lysine as a precursor must also be borne in mind) (9). Previous work with other systems has suggested several alternative pathways for pipecolate formation from lysine (22), but the results of the incorporation of labelled lysine into the pipecolate moiety of immunomycin (Fig. ​(Fig.1)1) clearly indicate loss of the α-nitrogen atom (3). More recently, the sequencing of the rap gene cluster revealed the presence of the rapL gene (Fig. ​(Fig.2),2), whose deduced gene product bears striking sequence similarity to two isoenzymes of ornithine deaminase from Agrobacterium tumefaciens (25, 26). Ornithine deaminase catalyzes the deaminative cyclization of ornithine to proline, and we have proposed (15) that the rapL gene product catalyzes the analogous conversion of l-lysine to l-pipecolate (Fig. ​(Fig.3).3). Open in a separate windowFIG. 2A portion of the rapamycin biosynthetic gene cluster which contains ancillary (non-polyketide synthase) genes (15, 27). PKS, polyketide synthase.Open in a separate windowFIG. 3(A) The conversion of l-ornithine to l-proline by ornithine cyclodeaminase (17). (B) Proposed conversion of l-lysine to l-pipecolic acid by the rapL gene product.Here, we report the use of ΦC31 phage-mediated gene replacement (10) to introduce a frameshift mutation into rapL and the ability of the mutant to synthesize rapamycins in the absence or presence of added pipecolate or pipecolate analogs.     

Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO “SimpleCell” (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.Most broad proteomic studies for discovery of cancer biomarkers in serum have been designed to interrogate the proteome and not taking into account that cancer cells often produce aberrant glycoforms (1). Many cancer biomarkers currently used in the clinic are based on circulating O-glycoproteins that are detected in established serological assays (CA125, CA15–3, CEA, and CA19.9) (2). In addition to being overexpressed in cancer, these proteins also carry aberrant glycans, which open for the opportunity to selectively detect aberrant glycoforms. An inherent problem with most cancer biomarker assays is that they often have poor specificity because the detected glycoprotein is found in elevated levels in nonmalignant conditions (2, 3). We recently found that the specificity of the widely used CA125 biomarker assay can be increased by selectively detecting aberrant O-glycoforms of the MUC16 mucin probed in the CA125 assay (4). Thus, the truncated O-glycan STn (NeuAcα2–6GalNAcα1-O-Ser/Thr)¹ (Fig. 1) was particularly suited for discrimination of MUC16 circulating in cancer patients in contrast to MUC16 circulating in benign conditions (4).Open in a separate windowFig. 1.Schematic depiction of the initial biosynthetic pathways of O-linked protein glycosylation. Overview of the O-linked protein glycosylation. O-GalNAc glycosylation is initiated by up to 20 different GalNAc-transferases. The addition of GalNAc to serines or threonines (or tyrosines) forms the Tn structure that can be sialylated by ST6GalNAc-I or further elongated to form up to four core structures. The core structures can be further elongated.One of the most characteristic phenotypes of cancer cells is the expression of truncated O-glycans, and the structures T (Galβ1–3GalNAcα1-O-Ser/Thr), STn, and Tn (GalNAcα1-O-Ser/Thr) (Fig. 1) are considered pancarcinoma antigens (2, 5). These truncated O-glycans are essentially not produced in normal and benign cells, which suggests that circulating O-glycoproteins in normal and benign conditions should have more mature O-glycans, whereas O-glycoproteins shed from cancer cells are expected to display truncated glycan structures. Cancer cells produce, secrete, and shed many different O-glycoproteins with truncated O-glycans, and provided these glycoproteins reach the circulation they may be detectable in serum. However, it is also known that nonsialylated glycoproteins are cleared from circulation through innate immune lectin receptors (6). In fact, we were previously unable to detect circulating T and Tn glycoforms of MUC1 and MUC16, while the sialylated ST (NeuAcα2–3Galβ1–3[NeuAcα2–6]_±GalNAcα1-O-Ser/Thr) and STn glycoforms were readily detectable (4, 7). Furthermore, two classical serological biomarker assays, CA19–9 (8) and CA72.4 (9–11), are based on the detection of sialylated O-glycans, and especially the latter that detects STn shows that proteins expressing the STn glycoform circulate in serum of cancer patients. Interestingly, although CA72.4 has been used for decades, it is still largely unknown which O-glycoproteins carry STn and are detected by the CA72.4 assay (9, 10).The truncated STn O-glycan has attracted much attention because it is highly expressed in most gastric (12), colorectal (13), ovarian (14), breast (15), pancreatic (16), and bladder (17) carcinomas, whereas expression of STn on normal tissues is highly restricted (11, 18). In addition, STn expression is associated with carcinoma aggressiveness and poor prognosis (15, 19). We have recently described the presence of a few STn bearing glycoproteins in serum from individuals with gastric cancer and gastric cancer precursor lesions (20). The biosynthetic and genetic mechanisms underlying the expression of this truncated O-glycan in cancer have remained poorly understood, and a number of mechanisms have been proposed that may not be mutually exclusive. One mechanism is the altered expression of the sialyltransferase ST6GalNAc-I, which is believed to be the main STn synthase (21, 22) (Fig. 1), and in fact overexpression of this enzyme in cell lines appears to override the normal O-glycan elongation machinery and result in expression of STn (22, 23). Another mechanism may be reduced core1 elongation that leads to accumulation of Tn, which serves as substrate for ST6GalNAc-I (22). The core1 synthase C1GALT1 is dependent on a private chaperone Cosmc, and several studies have reported that somatic mutations in COSMC gene (24), or hypermethylation of COSMC gene in cancer (25) lead to increased expression of Tn and STn. We have further shown that knockout (KO) of COSMC in a number of human cancer cell lines produce cells that express different levels of Tn and STn truncated O-glycans ranging from exclusive Tn to exclusive STn (26). A third potential mechanism offered recently may be related to cancer-associated relocation of the polypeptide GalNAc-transferases (GalNAc-Ts) that initiate O-glycosylation (Fig. 1) from Golgi to ER, which appear to induce expression of the Tn truncated O-glycans, although expression of STn has not been explored yet (27).In the present study, we applied a glycoproteomics strategy to explore potential biomarker O-glycoproteins with the STn glycoform in gastric cancer. We first characterized the O-glycoproteome and including the secretome of two gastric cancer cell lines, AGS (intestinal type gastric carcinoma) and MKN45 (diffuse type gastric carcinoma), using our SimpleCell (SC) discovery platform where we identified a total of 499 O-glycoproteins (1236 O-glycosites). This strategy involves genetic engineering of cell lines to produce homogenous truncated O-glycans (Tn and/or STn) by KO of COSMC, followed by Vicia Villosa lectin (VVA) enrichment of Tn glycoproteins and/or glycopeptides for sensitive identification of O-glycoproteins and O-glycosites by mass spectrometry (26, 28) (Fig. 1). We applied the same glycoproteomics workflow to a wild type (wt) gastric cancer cell line, KATO III (diffuse type gastric carcinoma), which naturally expresses Tn and STn O-glycans in a mixture with more complex structures, and identified a significantly smaller O-glycoproteome (total of 47 O-glycoproteins) compared with SimpleCells (total of 499 O-glycoproteins). We next modified the strategy to enrich for STn O-glycoproteins in pools of serum from cancer patients and normal controls using pretreatment with neuraminidase to remove sialic acid and expose Tn for VVA capture. This approach enabled us to isolate and identify 37 O-glycoproteins (49 O-glycosites) in gastric cancer serum. Finally, we confirmed that two of the identified serum O-glycoproteins (CD44 and GalNAc-T5) were expressed in gastric cancer tumors by immunohistology, and further used proximity ligation assay (PLA) to show that STn glycoforms of CD44 was expressed in cancer tissue. This study clearly shows that cancer patients have a variety of circulating O-glycoproteins with the STn glycoform, and supports the hypothesis that these glycoproteins originate from the cancer tissue. The identified secreted and circulating aberrant O-glycoproteins serve as a discovery set for biomarkers of gastric cancer.     

Limonene-1,2-Epoxide Hydrolase from Rhodococcus erythropolis DCL14 Belongs to a Novel Class of Epoxide Hydrolases

An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50°C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4′-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1,10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.Epoxides are highly reactive compounds which readily react with numerous biological compounds, including proteins and nucleic acids. Consequently, epoxides are cytotoxic, mutagenic, and potentially carcinogenic, and there is considerable interest in biological degradation mechanisms for these compounds.In bacteria, epoxides are formed during the metabolism of alkenes (23) and halohydrins (15, 26, 34, 49). Enzymes belonging to a large number of enzyme classes, including dehydrogenases (17), lyases (21), carboxylases (1, 43), glutathione S-transferases (6, 8), isomerases (24), and hydrolases (7, 19, 44), are involved in the microbial degradation of epoxides.Epoxide hydrolases are enzymes catalyzing the addition of water to epoxides forming the corresponding diol. This group of enzymes has been extensively studied in mammals, while only limited information is available on bacterial epoxide hydrolases. Three functions for epoxide hydrolases are recognized (42). In bacteria, epoxide hydrolases are involved in the degradation of several hydrocarbons, including 1,3-dihalo-2-propanol (34), 2,3-dihalo-1-propanol (15, 26), epichlorohydrin (46), propylene oxide (16), 9,10-epoxy fatty acids (30, 36), trans-2,3-epoxysuccinate (2), and cyclohexene oxide (14). Other epoxide hydrolases, such as microsomal and cytosolic epoxide hydrolase from mammals (for reviews, see references 4, 8, and 44), are involved in the detoxification of epoxides formed due to the action of P-450-dependent monooxygenases (8). Epoxide hydrolases are also involved in biosynthesis of hormones, such as leukotrienes and juvenile hormone (40, 45), and plant cuticular elements (11). Remarkably, the bacterial and eukaryotic epoxide hydrolases described so far form a homogeneous group of enzymes belonging to the α/β-hydrolase fold superfamily (10, 38).Rhodococcus erythropolis DCL14, a gram-positive bacterium, is able to grow on both (+)- and (−)-limonene as the sole source of carbon and energy (47). Cells grown on limonene contained a novel epoxide hydrolase that does not belong to the α/β-hydrolase fold superfamily. This limonene-1,2-epoxide hydrolase converts limonene-1,2-epoxide to limonene-1,2-diol (p-menth-8-ene-1,2-diol [Fig. 1]). In this report, we describe the purification and characterization of this enzyme and show that limonene-1,2-epoxide hydrolase belongs to a novel class of epoxide hydrolases. Open in a separate windowFIG. 1Reaction catalyzed by limonene-1,2-epoxide hydrolase.     

Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.