Biology subjects

Neupane, A.

Publications and source records attributed to Neupane, A..

6 recordsLinked to original sources

bioRxiv2026

High-resolution CRISPR/Cas9 screens identify PAK2 as a suppressor of macrophage proliferation

Macrophage abundance is regulated by pathways controlling survival and proliferation, yet the genetic determinants of macrophage fitness remain poorly understood. Here, we performed replicate genome-wide CRISPR/Cas9 screens in primary murine bone marrow-derived macrophages (BMDMs) to identify positive and negative regulators of macrophage fitness. Validity of the screens is supported by finding previously characterized core essential genes such as those encoding translation machinery, while disruption of established tumor suppressors increased BMDM representation in the culture. As expected, Csf1r encoding the macrophage growth factor emerged as a positive regulator of macrophage fitness. Unexpectedly, Pak2, encoding p21-activated kinase 2 (PAK2), emerged as a negative regulator of macrophage fitness. Targeted Pak2 disruption increased BMDM accumulation and was associated with elevated cyclin-D1 expression, identifying PAK2 as a suppressor of macrophage proliferation. PAK2 also localized to CSF1-induced actin-rich membrane ruffles, yet its depletion did not impair ruffle formation. Instead, PAK2-depleted macrophages exhibited persistent F-actin-rich ruffles, larger macropinosomes, and increased fluid-phase uptake. These changes were accompanied by altered LIMK/cofilin signaling and persistent cofilin localization at macropinocytic structures. Phosphoproteomic analysis further identified reduced phosphorylation of proteins associated with cytoskeletal regulation, phosphoinositide signaling, and endosomal trafficking. Together, these findings identify PAK2 as a context-dependent regulator that restrains macrophage proliferation and membrane-remodeling activity and demonstrate that PAK2 has markedly different fitness functions in primary macrophages than previously identified in several transformed cell types.

cell biology↗

bioRxiv2026

PI(3)P Signaling by VPS34 Complex II Orchestrates Macropinocytosis

Macrophage macropinocytosis contributes to wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Here, CRISPR/Cas9 whole-genome screens in murine bone-marrow derived macrophages (BMDM) identified genes regulating unstimulated, PMA-, and CSF1-stimulated uptake of the fluorescent pinocytosis solute tracer, Lucifer yellow. UVRAG and other members of VPS34 complex II (VPS34-II), which catalyze PI(3)P formation from phosphatidylinositol, were identified as positive regulators of macropinocytosis. Targeted gene disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with Uvrag-disrupted BMDM having fewer but larger macropinosomes. In contrast, depletion of ATG14, a unique component of VPS34 complex I, increased solute uptake and the number of macropinosomes. Live-cell imagining of macrophages expressing 2xFYVE-fluorescent protein fusions showed PI(3)P present on the plasma membrane, nascent macropinosomes, and endosomes. The presence of PI(3)P on the plasma membrane prior to macropinocytic cup closure, indicates a novel role on the plasma membrane. Quantitative imaging of fixed cells shows a decrease in concentration of PI(3)P in Uvrag-disrupted BMDMs while increase in concentration of PI(3)P in Atg14-disrupted BMDM compared to WT BMDMs. Treatment with the VPS34 inhibitor SAR-405 acutely decreased macropinocytosis but maintained AKT phosphorylation suggesting class I PI3K activity and PI(3,4,5)P3 production are independent of class III PI3K activity. These results suggest that PI(3)P is a key phosphoinositide governing macropinocytosis at the plasma membrane and that it is primarily formed via direct phosphorylation of PI rather than via the sequential dephosphorylation of PIP3.

cell biology↗

bioRxiv2026

Functional High Throughput Drug Screening Reveals Cyproheptadine as a Novel Treatment for LMNA -related Cardiomyopathy

ObjectivesTo define shared and variant-specific mechanisms underlying LMNA-associated dilated cardiomyopathy (DCM) and identify therapeutic candidates using human stem cell-based models. BackgroundVariants in the gene LMNA, encoding lamin A/C, cause 5-10% of dilated cardiomyopathies (DCM) and are strongly associated with heart failure and arrhythmias. Yet, the mechanisms by which LMNA variants drive disease and the distinction between shared and variant-specific phenotypes remain unclear. MethodsTo address this, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from six LMNA-DCM patients carrying three pathogenic variants (T150A, E381Afs*39, R527H) and from five healthy control patients. ResultsLMNA hiPSC-CMs exhibited nuclear membrane deformation, reduced beat rate, arrhythmias, and prolonged calcium transients. Transcriptomic and electrophysiological analyses revealed downregulation of cardiac genes and ion channels, with abnormal Ca{superscript 2} handling emerging as a shared disease mechanism. Leveraging a high-throughput functional assay, we performed an unbiased drug screen and identified cyproheptadine, an FDA-approved antihistamine, as the only compound to alleviate abnormal function across all LMNA variants. ConclusionOur findings reveal a shared disease mechanism across multiple LMNA variants driven by dysregulated Ca{superscript 2} handling. This work establishes a patient-specific drug discovery platform and identifies cyproheptadine as a promising therapeutic candidate for LMNA-associated dilated cardiomyopathy. HighlightsO_LIPatient-specific LMNA hiPSC-cardiomyocytes robustly recapitulate disease phenotypes, including nuclear defects, arrhythmias, and contractile dysfunction. C_LIO_LIDysregulated calcium handling emerges as a unifying mechanism driving pathology across distinct LMNA variants. C_LIO_LIVariant-resolved analysis reveals both shared and mutation-specific molecular and functional signatures. C_LIO_LIHigh-throughput screening identifies cyproheptadine as a potent, broadly effective rescue agent across all tested LMNA variants. C_LIO_LIThis work establishes a scalable precision medicine platform for rapid therapeutic discovery in inherited cardiomyopathies. C_LI

Cell Biology↗

bioRxiv2026

Lipolysis of host triacylglyceride-rich lipoproteins creates a toxic microenvironment for Staphylococcus aureus

Staphylococcus aureus secretes lipases that hydrolyze glycerol esters to mitigate host detection and scavenge fatty acids for membrane synthesis, but exposure to host-derived polyunsaturated fatty acids (PUFA) can be toxic. Here, we show that host hyperlipidemia protects against S. aureus osteomyelitis by engaging this vulnerability. Using a mouse model of severe hyperlipidemia, we show that PUFA-enriched plasma triacylglycerides are associated with reduced bacterial burden and bone destruction from injury-associated osteomyelitis. In vitro, human triacylglyceride-rich lipoproteins (TRL) exhibit potent bactericidal activity against post-exponential S. aureus that requires lipase activity of glycerol ester hydrolase (geh). Pre-exponential exposure to TRL suppressed agr quorum-sensing, blunting geh expression and toxicity; however, bactericidal activity of TRL could be restored by exogenous lipoprotein lipase. Together, these findings reveal TRL as active antimicrobial particles whose toxicity is unmasked by lipolysis and suggest that modulation of host lipid metabolism may provide new therapeutic opportunities for chronic S. aureus infection.

microbiology↗

bioRxiv2023

Structural and functional classification of G-quadruplex families within the human genome

G quadruplexes are short secondary DNA structures located throughout genomic DNA and transcribed RNA. though G4 structures have been shown to form in vivo, no current search tools are known to exist to examine these structures based on previously identified G quadruplexes, much less filter them based on similar sequence, structure, and thermodynamic properties. We present a framework for clustering G quadruplex sequences into families using the CD-HIT, MeShClust and DNACLUST methods along with a combination of Starcode and BLAST. Utilizing this framework to filter and annotate clusters, 95 families of G quadruplex sequences were identified within the human genome. Profiles for each family were created using hidden Markov models to allow for identification of additional family members and generate homology probability scores. The thermodynamic folding energy properties, functional annotation of genes associated with the sequences, scores from different prediction algorithms and transcription factor binding and motif to the G4 region for the sequences within a family were used to annotate and compare the diversity within and across clusters. The resulting set of G quadruplex families can be used to further understand how different regions of the genome are regulated by factors targeting specific structures common to members of a specific cluster.

bioinformatics↗

bioRxiv2023

Analysis of nucleotide variations in human g-quadruplex forming regions associated with disease states

While the role of G4 G quadruplex structures has been identified in cancers and metabolic disorders, single nucleotide variations (SNVs) and their effect on G4s in disease contexts have not been extensively studied. The COSMIC and CLINVAR databases were used to detect SNVs present in G4s to identify sequence level changes and their effect on alteration of G4 secondary structure. 37,515 G4 SNVs in the COSMIC database and 2,115 in CLINVAR were identified. Of those, 7,236 COSMIC (19.3%) and 416 (18%) of the CLINVAR variants result in G4 loss, while 2,728 (COSMIC) and 112 (CLINVAR) SNVs gain a G4 structure. The gene ontology term "GnRH (Gonadotropin-releasing hormone) secretion" is enriched in 21 genes in this pathway that have a G4 destabilizing SNV. Analysis of mutational patterns in the G4 structure show a higher selective pressure (3-fold) in the coding region on the template strand compared to the non-template strand. At the same time, an equal proportion of SNVs were observed among intronic, promoter and enhancer regions across strands. Using GO and pathway enrichment, genes with SNVs for G4 forming propensity in the coding region are enriched for Regulation of Ras protein signal transduction and Src homology 3 (SH3) domain binding.

genomics↗