Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, ...Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.
Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport ...activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from
(KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.
Polychlorinated biphenyls (PCBs) accumulate in mammals via the food chain because of their characteristics such as slow degradation and high hydrophobicity. One of the 209 PCB congeners, ...2,3',4,4',5-pentachlorobiphenyl (CB118), is abundantly found in the environment and in mammals. Understanding the metabolic fate of CB118 can provide important information toward evaluating its toxicity. In vitro studies on the metabolism of CB118 by cytochrome P450 enzymes (P450 or CYP) revealed that human CYP2B6 and rat CYP2B1 primarily metabolized it to 3-hydroxy (OH)-CB118, whereas rat CYP1A1 metabolized CB118 to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). Docking models of CYP2Bs with CB118 revealed a short distance between the 3-position of CB118 and the heme iron caused by polarization of the substrate-binding cavity, and maintenance of this pose through interaction with the peripheral amino acids determines the activity and position of hydroxylation. 4-Hydroxylation by rat CYP1A1 occurs owing to the longitudinal shape of the substrate-binding cavity toward the heme of CYP1A1. The metabolites 3-OH-CB118 and 4-OH-CB107 decreased potential for activating the aryl hydrocarbon receptor compared with that of CB118, thereby leading to a decrease in dioxin-like toxicity; however, the neurodevelopmental toxicity of 4-OH-CB107 has been previously reported. The results suggest that these 3 P450 isoforms play an important role in determining the extent of CB118 toxicity. This study will contribute to understanding of the metabolic fates and toxicity of CB118 in vivo.
Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and ...bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2′,3,6-tetrachlorobiphenyl (CB45) and 2,2′,3,4′,6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4′-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.
Cytochrome P450 (CYP) monooxygenases play critical roles in determining the toxicity of polychlorinated biphenyls (PCBs) in mammals. Hydroxylation of PCBs by these enzymes leads to increased water ...solubility, promoting the elimination of PCBs from the body. The CYP1 family is mainly responsible for metabolizing PCBs that exhibit a dioxin-like toxicity. Although the dioxin-like PCB 3,3′,4,4′-tetrachlorobiphenyl (CB77) is abundant in the environment and accumulates in organisms, information on CB77 metabolism by CYP1A1s is limited. In this study, recombinant rat CYP1A1 metabolized CB77 to 4′-hydroxy (OH)-3,3′,4,5′-tetrachlorobiphenyl (CB79) and 4′-OH-3,3′,4-trichlorobiphenyl (CB35), whereas human CYP1A1 produced only 4′-OH-CB79. Rat CYP1A1 exhibited much higher metabolizing activity than human CYP1A1 because CB77 was stably accommodated in the substrate-binding cavity of rat CYP1A1 and was close to its heme. In a rat CYP1A1 mutant with two human-type amino acids, the production of 4′-OH-CB79 decreased, whereas that of the dechlorinated metabolite 4′-OH-CB35 increased. These results are explained by a shift in the CB77 positions toward the heme. This study provides insight into the development of enzymes with high metabolizing activity and clarifies the structural basis of PCB metabolism, as dechlorination contributes to a drastic decrease in dioxin-like toxicity.
Display omitted
•Dioxin-like PCB CB77 was metabolized by rat and human CYP1A1s.•Hydroxylation and dechlorination were occurred in CB77 metabolism.•Stable accommodation of CB77 in the CYP1A1 cavity resulted in high activities.•Vicinity of CB77 to the CYP1A1 heme iron was accountable for the activity.•Two amino acids composing the substrate-binding cavity play critical roles.
Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation ...of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2′,3,4,4′,5′,6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (−)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (−)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3′–OH–CB183 and 5-OH-CB183, were identified. The docking study showed that the 3′-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.
Display omitted
•Atropisomers from racemic CB183 were used as substrate in metabolism experiments.•Atropisomer-dependent CB183 metabolism by human CYP2B6 was observed.•Stable accommodation of CB183 in the CYP2B6 cavity was significant for activity.•Vicinity of CB183 to the heme iron was also responsible for the activity.
Cytochromes P450 are major drug-metabolizing enzymes involved in the biotransformation of diverse xenobiotics and endogenous chemicals. Persistent organic pollutants (POPs) are toxic hydrophobic ...compounds that cause serious environmental problems because of their poor degradability. This calls for rational design of enzymes capable of catalyzing their biotransformation. Cytochrome P450 1A1 isoforms catalyze the biotransformation of some POPs, and constitute good starting points for the design of biocatalysts with tailored substrate specificity.
We rationalized the activities of wild type and mutant forms of rat cytochrome P450 1A1 towards 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and 3,3′,4,4′-tetrachlorobiphenyl (PCB77) using experiments and molecular dynamics simulations.
We showed that the enhanced activity of the CYP1A1 mutant towards TCDD was due to more efficient binding of the substrate in the active site even though the mutated site was over 2.5nm away from the catalytic center. Moreover, this mutation reduced activity towards PCB77.
Amino acids that affect substrate access channels can be viable targets for rational enzyme design even if they are located far from the catalytic site.
Display omitted
•The distal F240A mutation alters the hydroxylation activity of rat CYP1A1 towards two persistent organic pollutants (POPs) in opposing ways.•Both POPs have a high affinity for lipid membranes, from which they can enter the active site via channels capped by F/A 240.•The distal F240A mutation enabled efficient binding and positioning of TCDD in the CYP1A1 active site.•The F240A mutation destabilizes the binding of PCB77 by CYP1A1 and reduces the enzyme’s hydroxylation activity towards this substrate.•Both active site mutations and distal mutations can be exploited in rational enzyme design.
The present study investigated associations between epidemiological mumps patterns and meteorological factors in Japan. We used mumps surveillance data and meteorological data from all 47 prefectures ...of Japan from 1999 to 2020. A time-series analysis incorporating spectral analysis and the least-squares method was adopted. In all power spectral densities for the 47 prefectures, spectral lines were observed at frequency positions corresponding to 1-year and 6-month cycles. Optimum least-squares fitting (LSF) curves calculated with the 1-year and 6-month cycles explained the underlying variation in the mumps data. The LSF curves reproduced bimodal and unimodal cycles that are clearly observed in northern and southern Japan, respectively. In investigating factors associated with the seasonality of mumps epidemics, we defined the contribution ratios of a 1-year cycle (
) and 6-month cycle (
) as the contributions of amplitudes of 1-year and 6-month cycles, respectively, to the entire amplitude of the time series data.
and
were significantly correlated with annual mean temperature. The vaccine coverage rate of a measles-mumps-rubella vaccine might not have affected the 1-year and 6-month modes of the time series data. The results of the study suggest an association between mean temperature and mumps epidemics in Japan.
PDF
