Biology subjects

Doktor, T. K.

Publications and source records attributed to Doktor, T. K..

6 recordsLinked to original sources

bioRxiv2025

Acyl-CoA Binding Protein is Dispensable for White and Brown Adipose Tissue Function

Acyl-CoA binding protein (ACBP) plays a vital role in lipid metabolism by mediating the intracellular flux and utilization of long-chain acyl-CoAs. In this study we generated brown- and white adipose tissue specific knockout mice (Adipoq-Acbp-/-) and brown adipose tissue specific knockout mice (Ucp1-Acbp-/-) to investigate the role of ACBP in adipose tissue function. Here we demonstrate that loss of ACBP does not affect body weight, fat and lean mass, food intake and systemic energy expenditure, even under cold stress. Transcriptomic data show only minor changes in gene expression, whereas lipidomic profiling reveals a subtle increase in acyl-carnitines levels in brown adipose tissue. However, lipolytic activity in white adipose tissue as well as plasma glycerol, non-esterified fatty acid and triacylglycerol levels remained unaffected. In addition, no changes in mitochondrial respiration in BAT were observed. Taken together, our findings suggest that ACBP is dispensable for adipose tissue function and systemic energy metabolism, including thermoregulation.

biochemistry↗

bioRxiv2024

OLIGONUCLEOTIDES TARGETING THE SPLICE ACCEPTOR SITE DOWNSTREAM OF A MICROEXON AS AN INNOVATIVE THERAPY FOR AUTISM

BackgroundMicroexons are highly conserved and mostly neuronal-specific 3-27 nucleotide exons, enriched in genes linked to autism spectrum disorders (ASD). We have previously shown decreased inclusion of a neuronal specific 24 bp microexon (exon 4) of the translational regulator CPEB4 in brains of idiopathic ASD cases and that this leads to CPEB4 aggregation and subsequent under-expression of multiple high confidence ASD-risk genes. Furthermore, enhanced skipping of the CPEB4 microexon is also a novel etiological mechanism in schizophrenia (SCZ). MethodsIn this work we designed and tested in neuroblastoma cells a series of splice switching antisense oligonucleotides (SSO) targeting the CPEB4 genomic region surrounding the microexon. ResultsSSOs targeting candidate intronic regions near the microexon resulted in a decrease in microexon inclusion by blocking hnRNPC/PTPB1 binding, thus mimicking the isoform imbalance observed in ASD. However, based on the kinetic coupling model correlating transcriptional elongation with splicing regulation, we identified SSOs targeting downstream 3 splice site of exon 5 that favoured microexon inclusion in a dose-dependent manner and resulted in increased protein levels of FOXP1 and AUTS2, two high-confidence ASD risk genes that are known targets of CPEB4 and whose protein levels are reduced in ASD. ConclusionsThese results deepen our understanding of the complex splicing regulation of microexons and open new applications of SSOs to treat diseases such as ASD and SCZ that are characterized by altered microexon inclusion.

neuroscience↗

bioRxiv2023

Pan-cancer analysis of pyrimidine metabolism reveal signaling pathways connections with chemoresistance role

Deregulated nucleotide metabolism, and in particular increased pyrimidine metabolism (PyMet), has been shown to contribute to various pathological features of cancer including chemoresistance and epithelial-to-mesenchymal transition. However, cancer often encompasses complex signaling and metabolic pathway cascades for its progression, and understanding of these molecular regulatory processes in pyrimidine metabolism is quite limited. Therefore, a comprehensive pan-cancer analysis in around 10,000 gene expression profiles of 32 cancer types was employed using a pathway-based approach utilizing gene-sets representing various signaling and metabolic pathways. The analysis identified several top connections with PyMet including TERT, MTOR, DAX1, HOXA1, TP53 and TNC implying an inter-dependency of regulations which in turn was linked to the chemoresistance mechanisms. PyMet-signaling interactions were validated with in vitro derived gene-sets from endogenous thymidylate synthase (TYMS)-promoter activity reporter, from TYMS knockdown and from brequinar treatment, and further at single cell transcriptome level. Strikingly, brequinar treatment profile showed a strong inverse association pattern with doxorubicin chemoresistance in multiple cancer types. The study highlights the PyMet-pathway interactions and its role in chemoresistance, thereby providing an effective tool for improving PyMet targeting strategy in cancer. The analysis as an accessible resource is available at: www.pype.compbio.sdu.dk HighlightsPan-cancer analysis showed pyrimidine metabolism connections with signaling pathways Top pathway interactors of pyrimidine metabolism were TERT, HOXA1, TP53 and TNC In vitro derived pyrimidine gene-sets recapitulate cancer patients pathway analysis Pyrimidine associated pathways confer chemoresistance in multiple cancer types Pyrimidine metabolic inhibitor brequinar reversed doxorubicin chemoresistance feature

cancer biology↗

bioRxiv2023

Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia

Pseudoexons are nonfunctional intronic sequences that can be activated by deep intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder, propionic acidemia, by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. The PCCA pseudoexon can be exploited as a gene-regulatory switch, as healthy tissues show relatively high levels of inclusion. By blocking inclusion of the non-activated wild type pseudoexon, we increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels not only in patient fibroblasts harboring PCCA missense variants, but also those harboring PCCB missense variants. This could be a potential treatment strategy for propionic acidemia.

molecular biology↗

bioRxiv2023

All exons are not created equal - Exon vulnerability determines the effect of exonic mutations on splicing.

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements (SREs), but there is a misconception that all exons have an equal dependency on SREs and thus a similar vulnerability to aberrant splicing. We demonstrate that some exons are more likely to be affected by exonic splicing mutations (ESM) due to an inherent vulnerability, which is context-dependent and influenced by the strength of exon definition. We have developed VulExMap, a tool which based on empirical data that can designate whether a constitutive exon is vulnerable. Using VulExMap, we find that only 27% of all exons can be categorized as vulnerable whereas two-thirds of 332 previously reported ESMs in 71 disease genes are located in vulnerable exons. Because VulExMap analysis is based on empirical data on splicing of exons in their endogenous context, it includes all features important in determining the vulnerability. We believe that VulExMap will be an important tool when assessing the effect of exonic mutations by pinpointing whether they are located in exons vulnerable to ESMs.

molecular biology↗

bioRxiv2019

DeepCLIP: Predicting the effect of mutations on protein-RNA binding with Deep Learning

Nucleotide variants can cause functional changes by altering protein-RNA binding in various ways that are not easy to predict. This can affect processes such as splicing, nuclear shuttling, and stability of the transcript. Therefore, correct modelling of protein-RNA binding is critical when predicting the effects of sequence variations. Many RNA-binding proteins recognize a diverse set of motifs and binding is typically also dependent on the genomic context, making this task particularly challenging. Here, we present DeepCLIP, the first method for context-aware modeling and predicting protein binding to nucleic acids using exclusively sequence data as input. We show that DeepCLIP outperforms existing methods for modelling RNA-protein binding. Importantly, we demonstrate that DeepCLIP is able to reliably predict the functional effects of contextually dependent nucleotide variants in independent wet lab experiments. Furthermore, we show how DeepCLIP binding profiles can be used in the design of therapeutically relevant antisense oligonucleotides, and to uncover possible position-dependent regulation in a tissue-specific manner. DeepCLIP can be freely used at http://deepclip.compbio.sdu.dk.\n\nHighlightsO_LIWe have designed DeepCLIP as a simple neural network that requires only CLIP binding sites as input. The architecture and parameter settings of DeepCLIP makes it an efficient classifier and robust to train, making high performing models easy to train and recreate.\nC_LIO_LIUsing an extensive benchmark dataset, we demonstrate that DeepCLIP outperforms existing tools in classification. Furthermore, DeepCLIP provides direct information about the neural networks decision process through visualization of binding motifs and a binding profile that directly indicates sequence elements contributing to the classification.\nC_LIO_LITo show that DeepCLIP models generalize to different datasets we have demonstrated that predictions correlate with in vivo and in vitro experiments using quantitative binding assays and minigenes.\nC_LIO_LIIdentifying the binding sites for regulatory RNA-binding proteins is fundamental for efficient design of (therapeutic) antisense oligonucleotides. Employing a reported disease associated mutation, we demonstrate that DeepCLIP can be used for design of therapeutic antisense oligonucleotides that block regions important for binding of regulatory proteins and correct aberrant splicing.\nC_LIO_LIUsing DeepCLIP binding profiles, we uncovered a possible position-dependent mechanism behind the reported tissue-specificity of a group of TDP-43 repressed pseudoexons.\nC_LIO_LIWe have made DeepCLIP available as an online tool for training and application of proteinRNA binding deep learning models and prediction of the potential effects of clinically detected sequence variations (http://deepclip.compbio.sdu.dk/). We also provide DeepCLIP as a configurable stand-alone program (http://www.github.com/deepclip).\nC_LI

bioinformatics↗