bioRxiv Science⌕ Search

Biology subjects

Lee, H. B.

Publications and source records attributed to Lee, H. B..

7 recordsLinked to original sources

LoGoPPI enables fast and accurate protein protein interaction mapping at scale

Graph-based protein function analysis is powerful, but protein-protein interaction (PPI) networks exist for only a small fraction of animal and plant genomes. We present LoGoPPI, which infers PPIs from sequence by combining bi-encoder global protein representation with local residue-level late interaction. LoGoPPI matches or exceeds state-of-the-art PLM-based cross-encoders while achieving orders-of-magnitude faster inference, up to ~1,500-fold, and its local branch provides residue-level signals associated with interaction interfaces and structurally flexible regions. This efficiency enables practical proteome-wide PPI reconstruction at scales prohibitive for cross-encoder models, potentially extending interactome mapping to tens of thousands of animal and plant species. LoGoPPI thus provides a scalable framework for comparative and functional analysis of protein networks across diverse taxa.

bioinformatics↗

Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG carboxypeptidases DacC and DacA, in adaptation to alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline PG carboxypeptidase, whose enzyme activity and protein stability are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for the adaptation to salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all these roles of DacC and DacA were independent of LD-transpeptidases, which are necessary for the formation of PG 3-3 crosslinks and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs), DD-transpeptidases, mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of PG carboxypeptidases in stress adaptation and shape maintenance and provide novel insights into the cellular functions of PG carboxypeptidases associated with PBPs.

microbiology↗

The canonical HPA axis contributes to locomotion during photoadaptation but is not required

The hypothalamic-pituitary-adrenal (HPA) axis and its effector molecules--glucocorticoids--modulate diverse aspects of physiology in vertebrates ranging from metabolism to immune function, behavior, and circadian cycle. While the glucocorticoid receptor (nr3c1) is known to be involved in light adaptation (photoadaptation) of the retinal cells, the role of nr3c1 and other HPA axis signaling molecules in the behavioral phenotypes observed during photoadaptation have not been delineated. Therefore, we investigated locomotor adaptation to various light/dark durations using larval zebrafish that carry a mutated allele in key HPA axis receptors. First, we established baseline locomotion and compared WT and nr3c1 mutant larvae in constantly lit and dark conditions (for 12-hrs). WT larval zebrafish showed highest locomotor activity in the middle of the day and low activity during the early and later parts of the day, which was generally higher in the light. The baseline locomotion was depressed in nr3c1 mutants throughout the day in both environments with a more significant change noted in the light. Next, groups of larvae mutant in nr3c1, nr3c2 (mineralocorticoid receptor), or mc2r (melanocortin receptor type 2; adrenocorticotropic hormone (ACTH) receptor) along with their wildtype (WT) siblings were acclimated in the dark and underwent four cycles of dark-light illumination changes with different durations of illumination: 7.5, 6, 4, or 2 min. The nr3c1 and mc2r mutant fish showed significantly decreased locomotion during the dark phase when the illumination was provided for 4 or 2 min. However, with 4 min or longer illumination, if locomotor deficits were observed in mutant larvae, they demonstrated a "catch-up" phenotype with increasing locomotion during the later dark phases of the assay and ultimately reaching the swimming distances indistinguishable from their WT siblings in the assays with 7.5-min illumination. Finally, we looked at effects of light intensity, beginning with our short light exposure. A lower intensity of light failed to elicit any response even from WT fish after 1-min illumination. However, this dim light still evoked a robust response from nr3c1 mutants and their WT siblings after 7.5-min illumination. The nr3c2 mutant larvae consistently showed locomotor response similar to their WT siblings regardless of the length of the light phase. Thus, activation of the canonical HPA axis (i.e. nr3c1, mc2r) was necessary to induce a rapid locomotor adaptation following after light to dark transition with shorter light exposure times (~[≤]4 min), but it was not needed for locomotor responses observed after longer exposure to light (~> 4 min). Moreover, the locomotor response after transitioning to dark from a short (1 min) exposure was dependent on the intensity of light, whereas the longer exposure was not. Together, these findings suggest that locomotion after longer exposure to light is not dependent on the HPA axis, suggesting that either parallel independent pathway(s) are responsible for the locomotor response in these cases or that the HPA axis facilitates a primary pathway to increase sensitivity to light exposure.

neuroscience↗

Divergent LD-transpeptidase-independent effects of peptidoglycan carboxypeptidases on intrinsic ss-lactam and vancomycin resistance

Vancomycin and {beta}-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is D-alanine-D-alanine (D-Ala-D-Ala), whereas that of {beta}-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidases on vancomycin and {beta}-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of PG carboxypeptidases induced sensitivity to most {beta}-lactams, whereas it induced strong resistance toward vancomycin. Notably, both of two phenotypes did not have strong association with LD-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by increased amount of decoy D-Ala-D-Ala residues within PG, whereas {beta}-lactam sensitivity was associated with physical interactions between PG carboxypeptidase and PBPs. The presence of OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened {beta}-lactam sensitivity. Collectively, our results revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases, which involved inverse modulation of bacterial resistance to clinically important antibiotics, {beta}-lactams and vancomycin, and presented evidence for a link between PG carboxypeptidase and PBPs. IMPORTANCEBacterial peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. To date, most studies on PG carboxypeptidases are focused on LD-transpeptidase-related roles. We have revealed two distinct LD-transpeptidase-independent functions of PG carboxypeptidases with respect to antibiotic resistance. The deletion of PG carboxypeptidases led to sensitivity to most {beta}-lactams, while it caused strong resistance to vancomycin. The underlying molecular mechanisms of two phenotypes were not associated with LD-transpeptidases. Therefore, our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of {beta}-lactam antibiotics. One sentence summaryEffect of peptidoglycan carboxypeptidase on antibiotic

microbiology↗

Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5 or 3 junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5 or 3 junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

genetics↗

Glucocorticoid Regulation of Ependymal Glia and Regenerative Potential after Spinal Cord Injury

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, spinal cord tissue of vertebrate fish regenerates and restores function. Cord transection in zebrafish (Danio rerio) initially causes paralysis and neural cell death, with subsequent ependymal glial proliferation, extension of bipolar glia across the lesion, and neurogenesis. Axons extending from spared and nascent neurons along trans-lesional glial bridges restore functional connectivity. Here we report that glucocorticoids directly target the regeneration supporting changes in ependymal glia to inhibit neural repair. This effect is independent of hematogenic immune cells or microglia. Furthermore, glucocorticoid receptor signaling in ependymal glia is inversely regulated in rat models of spinal cord injury compared to zebrafish. The blockade of neural regeneration by glucocorticoids via a direct effect on ependymal glia has important clinical implications concerning the putative therapeutic benefit of corticosteroids in early management of spinal cord injury.

neuroscience↗

Locomotor response to acute stressors requires hypothalamic-pituitary-interrenal axis activation and glucocorticoid receptors in zebrafish

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response (SR). Rapid physiological changes in heart rate and blood sugar levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal (HPA) axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent HPA axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires HPI axis activation. In teleost fish, interrenal cells (I) are functionally homologous to the adrenal gland cortical layer. We derived 8 frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), two in each of exon 2 and exon 5 of nr3c1 (glucocorticoid receptor), and two in exon 2 of nr3c2 (mineralocorticoid receptor). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show here that this locomotor response requires a functioning HPI axis via the action of mc2r (adrenocorticotropic hormone receptor) and the canonical glucocorticoid receptor encoded by nr3c1 gene, but not mineralocorticoid receptor (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology may prove useful to screen for genetic, pharmacological, or environmental modifiers of the HPA axis.\n\nSignificanceAltered HPA axis activity is acknowledged as a causative and critical prognostic factor in many psychiatric disorders including depression. Nonetheless, genome wide association studies (GWAS) on depression have revealed conflicting findings about susceptibility loci, while identifying several genetic loci that warrant further investigations in the process. Such findings indicate that psychiatric disorders with complex genetic foundations require functional studies as well as genetic analyses. We developed a sensitive behavioral assay paradigm that leverages the genetic amenability and rapid development of zebrafish and demonstrated that our assay system reliably detects changes in HPA axis responsiveness. Our functional genetics and behavioral assay approach provides a useful platform to discover novel genetic, pharmacological, or environmental modifiers of the HPA axis.

neuroscience↗