SUMMARY Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.
Advances in genetic tools and sequencing technology in the past few years have vastly expanded our understanding of the genetics of neurodevelopmental disorders. Recent high-throughput sequencing analyses of structural brain malformations, cognitive and neuropsychiatric disorders, and localized cortical dysplasias have uncovered a diverse genetic landscape beyond classic Mendelian patterns of inheritance. The underlying genetic causes of neurodevelopmental disorders implicate numerous cell biological pathways critical for normal brain development.
Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible.
IEX-1 (Immediate Early response gene X-1) is a stress-inducible gene. It suppresses production of reactive oxygen species (ROS) and protects cells from apoptosis induced by a wide range of stimuli, but the underlying mechanism is not known. This study reveals that IEX-1 targets the mitochondrial F1Fo-ATPase Inhibitor (IF1) for degradation, resulting in acceleration of ATP hydrolysis, concomitant with reduction in ROS production. A prominent role for IF1 degradation in the function of IEX-1 was corroborated by siRNA-mediated gene silencing of IF1 that recapitulated the effects of IEX-1 on ATP hydrolysis and ROS production. Moreover, progressive C-terminal truncation studies demonstrated that IEX-1 interacted with the C terminus of IF1 and the interaction might render IF1 prone to degradation by an as yet unidentified mitochondrial protease. In support of a physiological importance of IEX-1 in the modulation of IF1 expression, gene-targeted deletion of IEX-1 stabilized IF1 and reduced mitochondrial F1Fo-ATPase activity in vivo. The altered activity of the F1Fo enzyme may account for a metabolic switch from oxidative phosphorylation toward glycolysis in IEX-1 deficient cells. Thus, IEX-1 deficient cells were more susceptible to glucose deprivation than wild type counterparts and displayed increased glucose uptake and lactate production in hypoxic conditions. The cells were also relatively refractory to oligomycin-mediated inhibition of ATP production. The studies offer novel insights into the primary role of IEX-1 in regulating a balance between energy provision and ROS production. Mitochondria are the site for the majority of cellular ATP synthesis, the main source for intracellular reactive oxygen species (ROS) production, and the key machinery for regulating cell death. These three biologic events are tied to the respiratory chain in the inner mitochondrial membrane. The electron transport chain consists of complex I, II, III, and IV, as well as an ATP synthase. The ATP synthase is comprised of the membrane-spanning Fo and the soluble F1 sectors, both of which are multiple protein complex.1,2 The F1Fo enzyme catalyzes the synthesis of ATP or oxidative phosphorylation utilizing the energy produced by the transmembrane electrochemical proton gradient along the respiratory chain. A high membrane potential (Dc m ) in the inner mitochondrial membrane as a consequence of cellular stress would slow the proton flux, giving rise to ROS formation. 3 Depending on the amounts of ROS generated, an intermediate level of ROS can amplify oxidant-sensitive signaling to promote cell proliferation, whereas too much of ROS production causes oxidative stress or mitochondrial membrane disruption that can trigger either apoptosis or necrosis. 4 Under conditions of oxygen deprivation, such as what occurs during ischemia or in the presence of an uncoupler of oxidative phosphorylation, the F1Fo-ATP synthase can switch from an ATP synthase to an ATPase, making it hydrolyze ATP produced in the cytosol by glycolysis. 5To preserve ATP, a nat...
Single-cell genomic analysis has grown rapidly in recent years and finds widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. To date, the amplification bias, amplification uniformity and reproducibility of the three major single cell whole genome amplification methods (GenomePlex WGA4, MDA and MALBAC) have not been systematically investigated using mammalian cells. In this study, we amplified genomic DNA from individual hippocampal neurons using three single-cell DNA amplification methods, and sequenced them at shallow depth. We then systematically evaluated the GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. The MALBAC displays significant biases towards high GC content. We then attempted to correct the GC bias issue by developing a bioinformatics pipeline, which allows us to call CNVs in single cell sequencing data, and chromosome level and sub-chromosomal level CNVs among individual neurons can be detected. We also proposed a metric to determine the CNV detection limits. Overall, MALBAC and WGA4 have better performance than MDA in detecting CNVs.
Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.
Remdesivir (RDV) exerts anti-severe acute respiratory coronavirus 2 activity following metabolic activation in the target tissues. However, the pharmacokinetics and tissue distributions of the parent drug and its active metabolites have been poorly characterized to date. Blood and tissue levels were evaluated in the current study. After intravenous administration of 20 mg/kg RDV in mice, the concentrations of the parent drug, nucleotide monophosphate (RMP) and triphosphate (RTP), as well as nucleoside (RN), in the blood, heart, liver, lung, kidney, testis, and small intestine were quantified. In blood, RDV was rapidly and completely metabolized and was barely detected at 0.5 h, similar to RTP, while its metabolites RMP and RN exhibited higher blood levels with increased residence times. The area under the concentration versus time curve up to the last measured point in time (AUC 0-t) values of RMP and RN were 4558 and 136,572 h•nM, respectively. The maximum plasma concentration (C max) values of RMP and RN were 2896 nM and 35,819 nM, respectively. Moreover, RDV presented an extensive distribution, and the lung, liver and kidney showed high levels of the parent drug and metabolites. The metabolic stabilities of RDV and RMP were also evaluated using lung, liver, and kidney microsomes. RDV showed higher clearances in the liver and kidney than in the lung, with intrinsic clearance (CL int) values of 1740, 1253, and 127 mL/(min•g microsomal protein), respectively.
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