Biology subjects

Karki, P.

Publications and source records attributed to Karki, P..

8 recordsLinked to original sources

bioRxiv2026

The Signaling Pathway of a Bacterial Two-Component System Determined by cryo-EM

Although two-component systems (TCSs) play ubiquitous roles in sensing signals for bacterial survival and virulence, a detailed structural map of the signal transduction pathway is lacking. Bacteriophytochromes (BphPs) are enzymatic photoreceptors with homologs in plants that regulate bacterial physiological responses via TCS signaling. Here, we employ cryo-EM to elucidate the structural basis of a light-regulated TCS by capturing the full-length BphP histidine kinase (HK) in complex with its downstream response regulator (RR). The RR binds as a dimer to the HK but contacts the enzyme only through one subunit. Light absorption perturbs the entire BphP structure, including the HK domain, which disrupts phosphorylation and phosphotransfer reactions and promote TCS complex dissociation. Thus, light controls signaling through both HK activity and reversible binding of the RR.

biophysics↗

bioRxiv2025

Adrenomedullin-RAMP2 enhances endothelial cell maturation synergistically with shear stress

Analysis of pulmonary vascular dysfunction in various lung pathologies remains challenging due to the lack of functional ex vivo models. Paracrine signaling in the lung plays a critical role in regulating endothelial maturation and vascular homeostasis. Previously, we employed single-cell RNA-sequencing (scRNAseq) to systematically map ligand-receptor (L/R) interactions within the lung vascular niche. However, the functional impact of these ligands on endothelial biology remained unknown. Here, we systematically evaluated selected ligands in vitro to assess their effects on endothelial barrier integrity, anti-inflammatory responses, and phenotypic maturation. Among the top soluble ligands, we found that adrenomedulin (ADM) exhibited superior barrier enhancing effect on human pulmonary endothelial cell monolayers, as evidenced by electrical cell impedance sensing (ECIS) and XperT assays. ADM also exhibited anti-inflammatory properties, decreasing ICAM1 and increasing IkBa expression in a dose-dependent manner. Shear stress (15 dynes/cm2) alone increased endothelial characteristics, including homeostatic markers such as CDH5, NOS3, TEK, and S1PR1. ADM treatment maintained the enhanced level of these markers under shear stress and further improved anti-coagulation by increasing THBD and decreasing F3 expression, and synergistically enhanced the expression of the native lung aerocyte capillary endothelial marker EDNRB. This effect was completely attenuated by a blockade of ADM receptor, RAMP2. Together, these findings identify ADM/RAMP2 signaling as a key paracrine pathway that enhances vascular barrier integrity, anti-inflammatory phenotype, and endothelial homeostasis, providing a framework for improving the physiological relevance of engineered vascular models.

bioengineering↗

bioRxiv2025

The Role of c-Jun Signaling in Cytidine Analog-Induced Cell Death in Melanoma

Melanoma stands as an increasingly pressing health concern. Enhanced mitochondrial metabolism has been reported in melanoma cells that survived treatment with traditional therapeutics, including cytidine analogs like gemcitabine (GEM). These findings suggest that chemotherapeutic drugs may play dual roles in promoting both cell survival and cell death, although the underlying mechanisms require further investigation. Herein, we conducted proteomics analysis on GEM-treated melanoma cells and found a drug-induced activation of DNA damage response and apoptosis, along with cell cycle arrest. Additionally, GEM treatment significantly altered protein networks related to mitochondrial ribosomal activity, the electron transport chain, and translation. Furthermore, we reported an upregulation of the JNK/c-Jun network in connection with the apoptotic proteins. Co-treatment with a Jun N-terminal Kinase (JNK) inhibitor, JNK-IN-8 (JNKi), significantly increased cell survival, suggesting the involvement of c-Jun signaling in GEM-induced cell death. Additionally, proteomics analysis revealed that JNKi downregulated apoptosis in co-treated cells, highlighting the potential role of the JNK/c-Jun network inhibition in chemotherapeutic tolerance. Collectively, our findings bridge gaps in understanding how melanoma cells respond to cytidine analogs by demonstrating the multifaceted effects of these agents in 1) inducing JNK-mediated apoptotic cell death, and 2) promoting a state of cell cycle inhibition.

cancer biology↗

bioRxiv2021

A Transient Metabolic State In Melanoma Persister Cells Mediated By Chemotherapeutic Treatments

Persister cells are defined as the small fraction of quiescent cells in a bulk cancer cell population that can tolerate unusually high levels of drugs. Persistence is a transient state that poses an important health concern in cancer therapy. The mechanisms associated with persister phenotypes are highly diverse and complex, and many aspects of persister cell physiology remain to be explored. We applied a melanoma cell line and panel of chemotherapeutic agents to show that melanoma persister cells are not necessarily preexisting dormant cells or stem cells; in fact, they may be induced by cancer chemotherapeutics. Our metabolomics analysis and phenotype microarray assays further demonstrated that the levels of Krebs cycle molecules are significantly lower in the melanoma persister subpopulation than in the untreated bulk cell population due to increased utilization rates in persisters. Our data indicate that this observed metabolic remodeling is transient, as the consumption rates of Krebs cycle metabolites are significantly reduced in the progenies of persisters. Given that the mitochondrial electron transport chain (ETC) is more active in the persister subpopulation than in the bulk cancer cell population, we also verified that targeting ETC activity can reduce melanoma persistence. The reported metabolic remodeling feature seems to be a conserved characteristic of melanoma persistence, as it has been observed in various melanoma persister subpopulations derived from a diverse range of chemotherapeutics. Elucidating a global metabolic mechanism that contributes to persister survival and reversible switching will ultimately foster the development of novel cancer therapeutic strategies.

cancer biology↗

bioRxiv2020

Tissue-specific regulation of translational readthrough tunes functions of the Traffic Jam transcription factor

Translational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR is pervasive in eukaryotic organisms; however, its biological significance remains unclear. In this study, we quantify the TR potential of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). We used CRISPR/Cas9 generated mutant flies to show that the TR-generated Tj isoform is expressed in the nuclei of a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads, which express the short Tj isoform only. Translational control of TR is critical for preservation of neuronal integrity and maintenance of reproductive health. Fine-tuning of the gene regulatory functions of transcription factors by TR provides a new potential mechanism for cell-specific regulation of gene expression. HighlightsO_LITj undergoes tissue-specific TR in neural cells of the central nervous system. C_LIO_LIStrict control of TR is crucial for neuroprotection and maintenance of reproductive capacity. C_LIO_LITR selectively fine-tunes the gene regulatory functions of the transcription factor. C_LIO_LITR in Tj links transcription and translation of tissue-specific control of gene expression. C_LI O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/411694v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1e6fb7dorg.highwire.dtl.DTLVardef@188d5c0org.highwire.dtl.DTLVardef@894173org.highwire.dtl.DTLVardef@127f4ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

Therapeutically Exploring Persister Metabolism In Bacteria

Bacterial persisters are rare phenotypic variants that are temporarily tolerant to high concentrations of antibiotics. We have previously discovered that persisters are mostly derived from stationary-phase cells with high redox activities that are maintained by endogenous protein and RNA degradation. This intracellular degradation resulted in self-inflicted damage that transiently repressed the cellular functions targeted by antibiotics. Leveraging this knowledge, we developed an assay integrating a degradable fluorescent protein system and a small library, containing FDA-approved drugs and antibiotics, to detect chemicals that target persister metabolism. We identified several metabolic inhibitors, including anti-psychotic drugs, that can reduce Escherichia coli persistence. These chemical inhibitors also reduce Pseudomonas aeruginosa persistence, potentially verifying the existence of similar mechanisms in a medically relevant organism.

microbiology↗

bioRxiv2019

INVESTIGATING THE EFFECTS OF OSMOLYTES AND ENVIRONMENTAL pH ON BACTERIAL PERSISTERS

Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance towards antibiotics. Persisters have been linked to the recalcitrance of biofilm related infections; hence, a complete understanding of the physiology of persisters can lead to improvement of therapeutic strategies associated with such infections. Mechanisms pertaining to persister formation are known to be related to stress response pathways triggered from intra- or extra-cellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH- induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, here we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was lessened in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

microbiology↗

bioRxiv2018

Dynamics of ribosomes and release factors during translation termination in E. coli

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

biochemistry↗