Studies directed towards a mechanistic evaluation of inactivation of aromatase by the suicide substrates androsta-1,4-diene-3,17-diones and its 6-ene derivatives aromatase inactivation by the 19-substituted derivatives and their enzymic aromatization

To gain insight into the mechanistic features for aromatase inactivation by the typical suicide substrates, androsta-1,4-diene-3,17-dione (ADD, 1) and its 6-ene derivative 2, we synthesized 19-substituted (methyl and halogeno) ADD and 1,4,6-triene derivatives 8 and 10 along with 4,6-diene derivatives 9 and tested for their ability to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. 19-Methyl-substituted steroids were the most powerful competitive inhibitors of aromatase (K_i: 8.2–40 nM) in each series. Among the 19-substituted inhibitors examined, 19-chloro-ADD and its 6-ene derivatives (7b and 9b) inactivated aromatase in a time-dependent manner in the presence of NADPH in air while the other ones did not. The time-dependent inactivation was blocked by the substrate AD and required NADPH. Only the time-dependent inactivators 7b and 9b in series of 1,4-diene and 1,4,6-triene steroids as well as all of 4,6-diene steroids 9, except for the methyl compound 9a, served as a substrate for aromatase to yield estradiol and/or its 6-ene estradiol with lower conversion rates compared to the corresponding parent steroids 1,4-diene, 1,4,6-triene and 4,6-diene derivatives. The present findings strongly suggest that the aromatase reaction, 19-oxygenation, at least in part, would be involved in the time-dependent inactivation of aromatase by the suicide substrates 1 and 2, where the 19-substitutent would play a critical role in the aromatase reaction probably though steric and electronic reasons.     

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

Evidence for expression of 11β-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis

11β-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP⁺-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP⁺-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP⁺-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.     

Biogeochemical Signals from Deep Microbial Life in Terrestrial Crust

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20–60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H₂ ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived ³He relative to radiogenic ⁴He in groundwater and the flux of H₂ along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H₂ from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Cross-talk between integrin α6β4 and insulin-like growth factor-1 receptor (IGF1R) through direct α6β4 binding to IGF1 and subsequent α6β4-IGF1-IGF1R ternary complex formation in anchorage-independent conditions

Integrin αvβ3 plays a role in insulin-like growth factor-1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk). The specifics of the cross-talk are, however, unclear. In a current model, "ligand occupancy" of αvβ3 (i.e. the binding of extracellular matrix proteins) enhances signaling induced by IGF1 binding to IGF1R. We recently reported that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation. Consistently, the integrin binding-defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, but it still binds to IGF1R. Like αvβ3, integrin α6β4 is overexpressed in many cancers and is implicated in cancer progression. Here, we discovered that α6β4 directly bound to IGF1, but not to R36E/R37E. Grafting the β4 sequence WPNSDP (residues 167-172), which corresponds to the specificity loop of β3, to integrin β1 markedly enhanced IGF1 binding to β1, suggesting that the WPNSDP sequence is involved in IGF1 recognition. WT IGF1 induced α6β4-IGF1-IGF1R ternary complex formation, whereas R36E/R37E did not. When cells were attached to matrix, exogenous IGF1 or α6β4 expression had little or no effect on intracellular signaling. When cell-matrix adhesion was reduced (in poly(2-hydroxyethyl methacrylate-coated plates), IGF1 induced intracellular signaling and enhanced cell survival in an α6β4-dependent manner. Also IGF1 enhanced colony formation in soft agar in an α6β4-dependent manner. These results suggest that IGF binding to α6β4 plays a major role in IGF signaling in anchorage-independent conditions, which mimic the in vivo environment, and is a novel therapeutic target.