Biology subjects

Loewen, C.

Publications and source records attributed to Loewen, C..

5 recordsLinked to original sources

bioRxiv2026

A Cell-Based Assay to Categorize Variants of Human Succinate Semialdehyde Dehydrogenase Associated with Autism Spectrum Disorder

Many genes are implicated in autism spectrum disorder (ASD) as a result of sequencing the genomes of individuals with ASD, however in most cases it remains unclear which genes are playing causative roles. ALDH5A1, which encodes the enzyme succinate semialdehyde dehydrogenase (SSADH), an important regulator of GABA metabolism, is causative in the syndrome SSADH deficiency and is also implicated in ASD. However, it is unknown how variants found in ASD affect SSADH function. We developed a yeast growth assay that models SSADH deficiency to quantify the functional impact of seven ALDH5A1 variants found in ASD. In this assay, expression of human ALDH5A1 partially complemented the growth defect caused by deletion of the ALDH5A1 ortholog UGA2. Using growth rate measurements, we calculated functional scores for 27 variants divided into calibration and ASD test variant groups. Functional scores for benign and pathogenic calibration variants segregated accordingly, validating the assay, while ASD variants displayed a range of activities from complete loss to normal function. Comparisons with published enzymatic assays and computational predictions showed broad agreement, while also identifying some limitations of these approaches. SUMMARY STATEMENTWe introduce a simple yeast assay to measure how autism-linked gene variants in ALDH5A1, which encodes the enzyme succinate semialdehyde dehydrogenase, affect its function, helping clarifying its role in autism.

neuroscience↗

bioRxiv2024

A multi-model approach defines function altering MECP2 missense variants identified in individuals with autism spectrum disorder

Pathogenic variants in MECP2 commonly lead to Rett syndrome, where MECP2s function as a DNA cytosine methylation reader is believed critical. MECP2 variants are also catalogued in individuals with autism spectrum disorder (ASD), including nine missense variants which had no known clinical significance at the start of this study. To assess these nine variants as risk alleles for ASD, we developed MECP2 variant functional assays using budding yeast and Drosophila. We calibrated these assays with known pathogenic and benign variants. Our data predict that four ASD variants are loss of function and five are functional. Protein destabilization offers insight into the altered function of some of these variants. Notably, yeast and Drosophila lack DNA methylation, yet all Rett pathogenic and ASD variants located in the methyl DNA binding domain that we analyzed proved to be loss of function, suggesting a clinically-relevant role for non-methyl DNA-binding by MECP2.

neuroscience↗

bioRxiv2024

PAN deadenylase is required for mitosis in response to microtubule stress in yeast and humans

HIGHLIGHTSO_LIPAN2-PAN3 catalytic activity is required for mitotic robustness under MT stress C_LIO_LICatalytic-dead Pan2-D1020A phenocopies pan2{Delta}: spindle defects and G2/M arrest C_LIO_LILoss of Pan2 catalytic activity amplifies nocodazole-induced changes in CLB1 and CLN3 mRNA C_LIO_LIPAN2 depletion in human cells produces multipolar spindles under MT stress C_LI IN BRIEFVerma et al. show that PAN2-PAN3 deadenylase catalytic activity is selectively required for mitotic fidelity under microtubule stress. Yeast pan2{Delta} cells exhibit spindle defects, G2/M arrest, and altered CLB1 and CLN3 mRNA levels under nocodazole. PAN2 knockdown in human cells produces multipolar spindles, demonstrating evolutionary conservation. Cytoplasmic deadenylation by PAN2-PAN3 and CCR4-NOT regulates mRNA stability and translation, but the contribution of individual deadenylases to mitotic progression is poorly defined. Here we show that PAN2-PAN3 catalytic activity is selectively required for mitotic robustness under microtubule stress. In budding yeast, the catalytically inactive Pan2-D1020A mutant fails to complement pan2{Delta} nocodazole sensitivity, leading to G2/M arrest, spindle defects, and increased cell death. pan2{Delta} exhibits synthetic genetic interactions with tubulin and tubulin-folding genes, and RNA-seq reveals dysregulation of cell-cycle transcripts including the cyclins CLB1 and CLN3. The phenotype is stress-conditional: pan2{Delta} cells proliferate normally without drug. In mammalian cells, PAN2 depletion sensitizes HEK293A cells to nocodazole and colchicine and produces prolonged metaphase with multipolar spindles. Together, these findings define a conserved, stress-conditional role for PAN2-PAN3 deadenylase activity in safeguarding mitotic fidelity when microtubules are compromised. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/591760v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19e6662org.highwire.dtl.DTLVardef@53ba18org.highwire.dtl.DTLVardef@de9c99org.highwire.dtl.DTLVardef@fb0f9d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO See accompanying graphical abstract file. C_FIG

cell biology↗

bioRxiv2023

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase, along with PMT1 or PMT2, two ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of Scj1p had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. ImportanceOur study showcases a newly developed high throughput screening technique to identify yeast mutant strains that exhibit an enhanced capacity for recombinant protein production. Using a genome-wide approach, we show that vesicle trafficking plays a crucial role in protein production, as the genes associated with this process are notably enriched in our screen. Furthermore, we demonstrate that a specific set of gene deletions, which were not previously recognized for their impact on recombinant laccase production, can be effectively manipulated in combination to increase the production of heterologous proteins. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

microbiology↗

bioRxiv2020

Sentinel Interaction Mapping (SIM) - A generic approach for the functional analysis of human disease gene variants using yeast

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using Synthetic Dosage Lethality screening, "sentinel" yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in high-throughput fashion through simple growth assays using either solid or liquid media. Sentinel Interaction Mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

genomics↗