Biology subjects

Lu, S. H.

Publications and source records attributed to Lu, S. H..

3 recordsLinked to original sources

bioRxiv2024

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

molecular biology↗

bioRxiv2021

Tankyrase interacts with the allosteric site of glucokinase and inhibits its glucose-sensing function in the beta cell

Insulin secretion in the pancreatic beta cell is rate-limited by glucokinase (GCK), the glucose sensor that catalyzes the first step of glucose metabolism. GCK consists of two lobes connected by a flexible hinge that allows the kinase to exhibit a spectrum of conformations ranging from the active, closed form to several inactive, less-compact forms. Activating GCK mutations can cause hyperinsulinemia and hypoglycemia in infants. A similar phenotype exhibited by tankyrase (TNKS)-deficient mice prompted us to investigate whether TNKS might modulate the glucose-sensing function of GCK. We found that TNKS colocalizes and directly interacts with GCK. Their interaction is mediated by two ankyrin-repeat clusters (ARC-2 and -5) in TNKS and a tankyrase-binding motif (TBM, aa 63-68) in the GCK hinge. This interaction is conformation sensitive, human GCK variants that cause hyperglycemia (V62M) or hypoglycemia (S64Y) enhance or diminish the interaction respectively, even though they have no impact on TNKS interaction in the context of a GCK peptide (V62M) or a peptide library (S64Y). Moreover, the TNKS-GCK interaction is inhibited by high glucose concentrations, which are known to stabilize GCK in the active (closed, glucose-avid) conformation. Conversely, glucose phosphorylation by GCK in vitro is inhibited by TNKS. To study this in vitro inhibitory effect in the MIN6 beta cells, we showed that glucose-stimulated insulin secretion is suppressed upon stabilization of the TNKS protein and is conversely enhanced upon TNKS knockdown. Based on these findings as well as by contrasting with hexokinase-2, we propose that TNKS is a physiological GCK inhibitor in pancreatic beta cells that acts by trapping the kinase in the open (inactive) conformation.

cell biology↗

bioRxiv2021

Comprehensive analysis of meiosis-derived cDNA libraries reveals gene isoforms and mitochondrial proteins important for competitive fitness

Gametogenesis is a highly regulated and dynamic developmental program where a diploid progenitor cell differentiates into haploid gametes, the precursors for sexual reproduction. During meiosis, several pathways converge to initiate ploidy reduction and organelle remodelling to render gametes competent for zygote formation and subsequent organismal development. Additionally, meiosis inherently rejuvenates the newly formed gametes resulting in lifespan resetting. Here, we construct five stage-specific, inducible meiotic cDNA libraries that represent over 84% of the yeast genome. We employ computational strategies to detect stage-specific meiotic transcript isoforms in each library and develop a robust screening pipeline to test the effect of each cDNA on competitive fitness. Our multi-day proof-of-principle time course reveals gene isoforms that are important for competitive fitness as well as mitochondrial proteins that cause dose-dependent disruption of respiration. Together, these novel meiotic cDNA libraries provide an important resource for systematically studying meiotic genes and gene isoforms in future studies. HIGHLIGHTSO_LIConstruction of five stage-specific, inducible meiotic cDNA libraries in budding yeast that collectively represent 5563 genes, which is over 84% of the genome C_LIO_LIAnalysis of the cDNA libraries reveal the presence of meiosis-specific transcript isoforms that are largely uncharacterized C_LIO_LIDevelopment of a robust gain-of-function screening pipeline identifies previously characterized genes and novel gene isoforms important for competitive fitness C_LIO_LIMulti-day proof-of-principle screen reveals mitochondrial proteins that cause dosage-specific respiration defects C_LI

genetics↗