Spotlight on CYP4B1

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy

Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells

AluY-mediated germline deletion, duplication and somatic stem cell reversion in <i>UBE2T</i> defines a new subtype of Fanconi anemia

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.</p

Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice

Short-Term Audiovisual Spatial Training Enhances Electrophysiological Correlates of Auditory Selective Spatial Attention

Audiovisual cross-modal training has been proposed as a tool to improve human spatial hearing. Here, we investigated training-induced modulations of event-related potential (ERP) components that have been associated with processes of auditory selective spatial attention when a speaker of interest has to be localized in a multiple speaker (“cocktail-party”) scenario. Forty-five healthy participants were tested, including younger (19–29 years; n = 21) and older (66–76 years; n = 24) age groups. Three conditions of short-term training (duration 15 min) were compared, requiring localization of non-speech targets under “cocktail-party” conditions with either (1) synchronous presentation of co-localized auditory-target and visual stimuli (audiovisual-congruency training) or (2) immediate visual feedback on correct or incorrect localization responses (visual-feedback training), or (3) presentation of spatially incongruent auditory-target and visual stimuli presented at random positions with synchronous onset (control condition). Prior to and after training, participants were tested in an auditory spatial attention task (15 min), requiring localization of a predefined spoken word out of three distractor words, which were presented with synchronous stimulus onset from different positions. Peaks of ERP components were analyzed with a specific focus on the N2, which is known to be a correlate of auditory selective spatial attention. N2 amplitudes were significantly larger after audiovisual-congruency training compared with the remaining training conditions for younger, but not older, participants. Also, at the time of the N2, distributed source analysis revealed an enhancement of neural activity induced by audiovisual-congruency training in dorsolateral prefrontal cortex (Brodmann area 9) for the younger group. These findings suggest that cross-modal processes induced by audiovisual-congruency training under “cocktail-party” conditions at a short time scale resulted in an enhancement of correlates of auditory selective spatial attention

Effects of age on electrophysiological correlates of speech processing in a dynamic “cocktail-party” situation

Successful speech perception in multi-speaker environments depends on auditory scene analysis, comprising auditory object segregation and grouping, and on focusing attention toward the speaker of interest. Changes in speaker settings (e.g., in speaker position) require object re-selection and attention re-focusing. Here, we tested the processing of changes in a realistic multi-speaker scenario in younger and older adults, employing a speech-perception task, and event-related potential (ERP) measures. Sequences of short words (combinations of company names and values) were simultaneously presented via four loudspeakers at different locations, and the participants responded to the value of a target company. Voice and position of the speaker of the target information were kept constant for a variable number of trials and then changed. Relative to the pre-change level, changes caused higher error rates, and more so in older than younger adults. The ERP analysis revealed stronger fronto-central N2 and N400 components in younger adults, suggesting a more effective inhibition of concurrent speech stimuli and enhanced language processing. The difference ERPs (post-change minus pre-change) indicated a change-related N400 and late positive complex (LPC) over parietal areas in both groups. Only the older adults showed an additional frontal LPC, suggesting increased allocation of attentional resources after changes in speaker settings. In sum, changes in speaker settings are critical events for speech perception in multi-speaker environments. Especially older persons show deficits that could be based on less flexible inhibitory control and increased distraction

Reciprocal modulation of Aβ42 aggregation by copper and homocysteine

Hyperhomocysteinemia is a risk factor for Alzheimer's disease (AD). Both homocysteine (Hcy) and amyloid β (Aβ), which accumulates in the brain of AD patients, bind copper. Aim of this study was to test the hypothesis that the association of Hcy and AD results from a molecular interaction between Hcy and Aβ that is mediated by copper. We established a microtiter plate format thioflavin T aggregation assay to monitor Aβ42 fibrillization. Copper (5 μM) completely prevented Aβ42 (5 μM) fibrillization. Homocysteine in the absence of copper did not impact Aβ42 fibrillization, but physiological concentrations of Hcy (10-100 μM) attenuated the inhibitory effect of copper on Aβ42 fibril formation. These results were qualitatively confirmed by electron microscopy, which did not reveal morphological differences. To compare the toxicity of fibrillar and non-fibrillar Aβ42 exposed to copper or Hcy, rat primary cortical neurons were treated in vitro with 5 μM Aβ42 for 72 h. After incubation with 5 μM Aβ42 that had been aggregating in the absence of Hcy or copper, cell viability was reduced to 40%. Incubation with 5 μM Aβ42, in which fibril formation had been prevented or reverted by the addition of 5 μM copper, resulted in cell viability of approximately 25%. Accordingly, viability was reduced to 25% after incubation with 5 μM monomeric, i.e., non-fibrillized, Aβ42. The addition of Hcy plus copper to 5 μM Aβ42 yielded 50% viability. In conclusion, copper prevents and reverts Aβ fibril formation leading rather to formation of lower order oligomers or amorphous aggregates, and Hcy reduces these effects. Such mechanisms may explain the association of hyperhomocysteinemia and AD, leading to novel therapeutic strategies in the prevention and treatment of this disease