Biology subjects

Old, W. M.

Publications and source records attributed to Old, W. M..

6 recordsLinked to original sources

bioRxiv2020

Multi-Omic Analysis Reveals Cannabidiol Disruption of Cholesterol Homeostasis in Human Cell Lines

The non-psychoactive cannabinoid, cannabidiol (CBD), is FDA-approved for treatment of two drug-resistant epileptic disorders, and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multi-omic profiling. FRET-based biosensor screening revealed that CBD treatment resulted in a sharp rise in cytosolic calcium, and activation of AMPK and ERK kinases in human keratinocyte and neuroblastoma cell lines. CBD treatment led to alterations in the abundance of metabolites, mRNA transcripts, and proteins consistent with activation of cholesterol biosynthesis, transport and storage. We found that CBD rapidly incorporated into cellular membranes and altered cholesterol chemical activity, suggesting direct perturbation of cholesterol-dependent membrane properties. CBD treatment induced apoptosis in a dose-dependent manner in multiple human cell lines, which was rescued by inhibition of cholesterol synthesis, and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

systems biology↗

bioRxiv2019

Mutations that improve the efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

New enzymes often evolve by amplification and divergence of genes encoding enzymes with a weak ability to provide a new function. Experimental studies to date have followed the evolutionary trajectory of an amplified gene, but have not addressed other mutations in the genome when fitness is limited by an evolving gene. We have adapted Escherichia coli in which an enzymes weak secondary activity has been recruited to serve an essential function. While the gene encoding the \"weak-link\" enzyme amplified in all eight populations, mutations improving the new activity occurred in only one. This beneficial allele quickly swept the amplified array, displacing the parental allele. Most adaptive mutations, however, occurred elsewhere in the genome. We have identified the mechanisms by which three of the classes of mutations increase fitness. These mutations may be detrimental once a new enzyme has evolved, and require reversion or compensation, leading to permanent changes in the genome.

evolutionary biology↗

bioRxiv2019

Rapid discovery of drug target engagement by isothermal shift assay

Identifying protein targets directly bound by drug molecules within living systems remains challenging. Here we present the isothermal shift assay, iTSA, for rapid identification of drug targets. Compared with thermal proteome profiling, a prevailing method for target engagement, iTSA offers a simplified workflow, 4-fold higher throughput, and multiplexed experimental designs with higher replication. We demonstrate application of iTSA to identify targets for several kinase inhibitors in lysates and living cells.

pharmacology and toxicology↗

bioRxiv2018

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

Loss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known. Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus.

systems biology↗

bioRxiv2018

Cerebral organoid proteomics reveals signatures of dysregulated cortical development associated with human trisomy 21

Human trisomy 21 (Down syndrome) is the most common genetic cause of intellectual disability, and is associated with complex perturbations in protein expression during development. Brain region-specific alterations in neuronal density and composition originate prenatally in trisomy 21 individuals, and are presumed to underlie the intellectual disability and early onset neurodegeneration that characterizes Down syndrome. However, the mechanisms by which chromosome 21 aneuploidy drives alterations in the central nervous system are not well understood, particularly in brain regions that are uniquely human and thus inaccessible to established animal models. Cerebral organoids are pluripotent stem cell derived models of prenatal brain development that have been used to deepen our understanding of the atypical processes associated with human neurobiological disorders, and thus provide a promising avenue to explore the molecular basis for neurodevelopmental alterations in trisomy 21. Here, we employ high-resolution label-free mass spectrometry to map proteomic changes over the course of trisomy 21 cerebral organoid development, and evaluate the proteomic alterations in response to treatment with harmine, a small molecule inhibitor of the chromosome 21 encoded protein kinase DYRK1A. Our results reveal trisomy 21 specific dysregulation of networks associated with neurogenesis, axon guidance and extracellular matrix remodeling. We find significant overlap of these networks show significant overlap with previously identified dysregulated gene expression modules identified in trisomy 21 fetal brain tissue. We show that harmine leads to partial normalization of key regulators of cortical development, including WNT7A and the transcription factors TBR1, BCL11A, and POU3F2, pointing to a causative role for DYRK1A over-expression in neurodevelopmental effects of human trisomy 21.

systems biology↗

bioRxiv2017

Identification of the primary peptide inhibitor contaminant of fibrillation and toxicity in synthetic Amyloid-β42

Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the A{beta}42{Delta}39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable A{beta}42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimers research in particular, and biological research in general.

neuroscience↗