Biology subjects

Najar, M. A.

Publications and source records attributed to Najar, M. A..

13 recordsLinked to original sources

bioRxiv2026

Systems-level proteomic reprogramming reveals mitochondrial restoration and inhibition of Rho GTPase-mediated cytoskeletal and inflammatory signaling in CKD

Chronic kidney disease (CKD) is a progressive disorder characterized by metabolic dysfunction, mitochondrial impairment, oxidative stress, and chronic inflammation, ultimately leading to irreversible renal damage. Despite advances in understanding CKD pathophysiology, effective therapies targeting these interconnected molecular processes remain limited. In this study, we performed a comprehensive data-independent acquisition (DIA)-based proteomic analysis to investigate the molecular alterations associated with CKD and to evaluate the therapeutic impact of DVA treatment. Using a CKD model with three treatment conditions (DVA, KY, and DVA+KY) alongside disease and healthy controls, we quantified global proteomic changes and applied statistical filtering (fold change [≥]2, p [≤]0.05) followed by K-means clustering (k=10). Distinct protein clusters revealed bidirectional modulation upon DVA treatment. Notably, Cluster 1 comprised proteins downregulated in CKD but significantly restored following DVA administration, while Cluster 2 included proteins elevated in CKD that were suppressed by DVA. Pathway enrichment and network analyses demonstrated that Cluster 1 proteins were predominantly associated with mitochondrial function, oxidative phosphorylation, and metabolic processes, whereas Cluster 2 proteins were enriched in immune signaling, oxidative stress, cytoskeletal remodeling, and proteostasis pathways. At the molecular level, DVA treatment restored key mitochondrial and metabolic regulators, including components of the electron transport chain (e.g., COX5A, NDUFS5, SDHB) and redox homeostasis proteins, indicating recovery of cellular bioenergetics. Concurrently, DVA suppressed inflammatory mediators (STAT2, IFI47, GBP2), oxidative stress-related proteins (CYBB, PRDX5), and cytoskeletal regulators linked to renal injury (ARHGEF12, FMNL2). Network and Reactome analyses further confirmed coordinated modulation of interconnected biological systems rather than isolated protein changes. Collectively, our findings demonstrate that DVA exerts a dual therapeutic effect by restoring essential mitochondrial and metabolic pathways while simultaneously suppressing inflammation, oxidative stress, and cytoskeletal dysregulation in CKD. This systems-level proteomic reprogramming highlights DVA as a promising candidate for CKD intervention and provides mechanistic insights into disease progression and therapeutic targeting.

systems biology↗

bioRxiv2026

Temporal phosphoproteomics reveals rapid restoration of kinase signaling by Glycyrrhiza glabra in a rotenone-induced Parkinson disease model

Parkinsons disease is a progressive neurodegenerative disorder associated with mitochondrial dysfunction, oxidative stress, impaired autophagy, and dysregulated cellular signaling pathways. Although Glycyrrhiza glabra has been reported to exhibit neuroprotective properties, the early phosphorylation-mediated signaling mechanisms underlying its protective effects remain poorly understood. In this study, we employed a Tandem Mass Tag (TMT)-based temporal quantitative phosphoproteomic approach to investigate early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection in a rotenone-induced in vitro PD model. Differentiated IMR-32 neuronal cells were treated with rotenone alone or in combination with Glycyrrhiza glabra extract, and phosphoproteomic alterations were analyzed at 2, 5, 15, and 30 minutes using liquid chromatography coupled with tandem mass spectrometer. Temporal phosphoproteomic analysis identified 6,424 phosphopeptides corresponding to 2,368 phosphoproteins and 5,468 phosphorylation sites. Comparative analysis revealed extensive phosphorylation rewiring induced by rotenone and restoration of several dysregulated phosphorylation events following Glycyrrhiza glabra co-treatment. More than 130 phosphoproteins and multiple kinase-associated signaling pathways were dynamically regulated across the temporal conditions. Kinase enrichment analysis identified restoration of several critical kinases, including AKT1, MTOR, MAPK1/3, PRKACA, PRKCD, and GSK3A/B, which are associated with neuronal survival, stress adaptation, and autophagy. Integrated pathway and kinase-substrate interaction analyses further revealed enrichment of AMPK signaling, FOXO signaling, receptor tyrosine kinase signaling, RNA processing, and cell-cycle regulatory pathways. Notably, several spliceosome-associated phosphoproteins demonstrated dynamic phosphorylation changes during the early neuroprotective response. Collectively, this study provides a detailed temporal phosphoproteomic landscape of early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection and highlights kinase-driven signaling pathways that may represent potential therapeutic targets in Parkinsons disease.

neuroscience↗

bioRxiv2026

Extraction-dependent bone proteomics reveals distinct stable and dynamic protein modules during early post-exposure degradation

Bone is a highly durable biological tissue widely used in forensic, archaeological, and anthropological investigations; however, efficient protein recovery and understanding of protein stability over time remain major challenges in skeletal proteomics. Here, we systematically evaluated three bone protein extraction workflows and integrated them with data-independent acquisition (DIA) mass spectrometry to assess proteome coverage, reproducibility, and temporal protein dynamics under environmentally exposed conditions. Comparative analysis demonstrated that extraction strategy is a primary determinant of detectable proteome composition. EDTA-based demineralization followed by SDS extraction provided the deepest proteome coverage and highest reproducibility, whereas guanidine hydrochloride extraction preferentially enriched collagen and extracellular matrix proteins. In contrast, acid-based extraction yielded limited protein recovery. Temporal profiling of bone samples collected at 10 and 45 days post-exposure revealed two distinct protein classes. A temporally stable module, enriched in collagens and extracellular matrix proteins including COL1A2, COL5A2, BGN, SPARCL1, and NID2, exhibited minimal abundance change, indicating resistance to environmental degradation. In contrast, temporally dynamic proteins, enriched in mitochondrial, metabolic, and intracellular pathways such as ACO2, OGDH, PDHA1, ATP5PO, and PFKM, showed marked decline over time. These findings support a two-compartment model of bone protein preservation in which matrix-embedded proteins are preferentially retained while exposed intracellular proteins undergo progressive degradation. Collectively, this study establishes an integrated framework linking extraction methodology with temporal proteome stability and identifies candidate markers for skeletal preservation assessment and temporal biomarker development in forensic and archaeological applications.

systems biology↗

bioRxiv2026

Integrated Metabolomic, Proteomic, and Phosphoproteomic Profiling Reveals CAMKK2-Dependent Regulation of Cell Cycle and Nucleotide Metabolism in Gastric Cancer

Gastric cancer is driven by aberrant kinase signaling that supports uncontrolled proliferation and metabolic adaptation. Calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, its role in regulating metabolic programs that sustain tumor growth remains incompletely understood. In this study, we employed an integrated multi-omics approach with a primary focus on untargeted metabolomics to investigate the consequences of CAMKK2 inhibition in gastric cancer cells. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells resulted in significant suppression of proliferation, clonogenic growth, migration, and invasion, accompanied by pronounced nuclear abnormalities and multinucleation indicative of mitotic defects. Global metabolomic profiling revealed extensive and time-dependent metabolic reprogramming following CAMKK2 inhibition, characterized by a marked depletion of nucleotide intermediates, including purine and pyrimidine metabolites required for DNA synthesis. Pathway enrichment analysis highlighted suppression of nucleotide metabolism, lipid metabolism, and central carbon metabolic pathways, indicating a broad impairment of biosynthetic capacity. Integration with proteomic and phosphoproteomic datasets demonstrated that metabolic alterations were accompanied by downregulation of DNA replication machinery and attenuation of kinase signaling pathways governing cell cycle progression. Protein metabolite interaction and docking analyses further supported functional coupling between nucleotide metabolites and key replication-associated enzymes, revealing disruption of metabolite enzyme interactions upon CAMKK2 inhibition. Collectively, these findings identify CAMKK2 as a critical regulator of metabolic programs that support DNA replication and cell cycle progression. Its inhibition induces replication stress through coordinated depletion of nucleotide pools and disruption of replication-associated signaling, leading to impaired proliferation and mitotic failure. These results highlight CAMKK2 as a potential therapeutic target for exploiting metabolic vulnerabilities in gastric cancer.

cancer biology↗

bioRxiv2026

Systems level phosphoproteomics reveals CAMKK2 driven kinase signaling underlying malignant phenotypes in gastric cancer

Gastric cancer is driven by aberrant kinase signaling that promotes uncontrolled proliferation and malignant progression. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, the global phosphorylation networks downstream of CAMKK2 remain incompletely defined. In this study, we investigated the functional and signaling consequences of CAMKK2 inhibition in gastric cancer cells using an integrated phenotypic and quantitative phosphoproteomics approach. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells significantly suppressed proliferation, clonogenic growth, migration, and invasion, and induced defects in nuclear morphology indicative of impaired cell cycle progression. Tandem mass tag (TMT) based phosphoproteomic profiling identified over 10,500 phosphopeptides and revealed extensive phosphoproteome remodeling following CAMKK2 inhibition, characterized predominantly by hypophosphorylation of proteins involved in nuclear signaling, RNA processing, and cell cycle regulation. Kinase substrate enrichment and motif analyses demonstrated coordinated attenuation of CDK, MAPK, and mitotic kinase-associated signaling pathways, with convergence on E2F regulated transcriptional programs. Collectively, these findings establish CAMKK2 as a central regulator of kinase signaling networks that sustain proliferative and malignant phenotypes in gastric cancer and highlight CAMKK2 inhibition as a potential therapeutic strategy.

systems biology↗

bioRxiv2026

Phosphoproteomic profiling reveals signaling pathways modulated by Annona muricata leaf extract in oral adenosquamous carcinoma cells

Phosphorylation driven dysregulation of intracellular signaling networks is a central feature of cancer initiation, progression, and therapeutic resistance. Although Annona muricata leaf extracts have demonstrated anticancer activity across multiple experimental models, the underlying molecular mechanisms particularly at the level of phosphorylation dependent signaling remain poorly understood. In this study, we employed a tandem mass tag TMT-based quantitative phosphoproteomic approach to systematically characterize signaling alterations induced by methanolic Annona muricataleaf extract (AME) in oral squamous cell carcinoma (OSCC) CAL-27 cells. Functional assays revealed that AME significantly inhibited cell proliferation, migration, and clonogenic survival. Global phosphoproteomic profiling identified 6,362 phosphopeptides corresponding to 1,964 unique phosphorylation sites across nearly 7,000 phosphoproteins. AME treatment induced widespread, time-dependent hypophosphorylation, indicating a coordinated suppression of oncogenic signaling networks. Pathway and network analyses revealed marked inhibition of signaling pathways associated with key oncogenic kinases, including cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), and signaling modules linked to EGFR and mTOR pathways. Kinase-substrate enrichment and kinome mapping further demonstrated reduced inferred activity of CDK and MAPK driven signaling, accompanied by suppression of cell cycle progression, mitosis, and checkpoint regulation. Collectively, these findings demonstrate that AME induces systems-level remodeling of phosphorylation dependent signaling networks, enforcing a growth-restrictive cellular state in OSCC cells. This study highlights quantitative phosphoproteomics as a powerful strategy for dissecting natural product mediated regulation of oncogenic signaling and provides mechanistic insight into the anticancer potential of Annona muricata.

cancer biology↗

bioRxiv2026

Serial enrichment based quantitative proteomics enables deep and multiplexed profiling of post-translational modifications

Post-translational modifications (PTMs) play central roles in regulating protein function, localization, stability, and signaling networks. Comprehensive characterization of multiple PTMs, however, remains technically challenging due to limited sample availability, enrichment, incompatibilities, and analytical complexity. Here, we present an integrated serial enrichment workflow that enables quantitative profiling of multiple PTMs from a single biological sample. Using a streamlined strategy that combines StageTip-based fractionation with sequential immunoaffinity and metal oxide affinity enrichment, we achieved robust identification and quantification of lysine acetylation, lysine succinylation, phosphotyrosine, and global phosphorylation within the same sample. Application of this workflow resulted in deep proteome coverage alongside high-confidence PTM site identification with minimal sample loss and high reproducibility. Importantly, serial enrichment preserved PTM specificity and enabled comparative quantitative analysis across modification types. This approach provides a practical and scalable solution for integrated multi-PTM analysis, facilitating comprehensive interrogation of proteome regulation under limited sample conditions and offering broad applicability to systems biology, disease profiling, and translational proteomics studies.

systems biology↗

bioRxiv2025

Coordination of nuclear RNA processing by speckle-localized kinase TAOK2

Nuclear speckles are membraneless organelles that act as active splicing hubs especially at sites of high transcription. Emerging views of this dynamic subnuclear structure place it as a hub of RNA processing, impacting steps from transcription to export. To manage this complex microcosm of RNA metabolism, nuclear speckles also require kinases and phosphorylation to execute their functions. The nuclear speckle-localized kinase, TAOK2, mediates the splicing and export of viral transcripts at the nuclear speckle, but its role in the processing of cellular transcripts was unknown. We used siRNA knockdown of TAOK2 and assessed RNA transcripts in both whole cell and nucleocytoplasmic fractions to characterize the complete endogenous effects of TAOK2. We found that TAOK2 knockdown impacts over 10% of the transcriptome, through changes in alternative splicing, export and transcript abundance. Cellular and biochemical phosphoproteomics further revealed nuclear speckle scaffolding proteins SRRM1 and SRRM2 as potential direct phosphorylation targets of TAOK2, mediating its large effects on speckle integrity and speckle-localized splicing. Indeed, knockdown of TAOK2 perturbs almost all speckle-resident serine/arginine (SR)-rich proteins while leaving heterogeneous ribonucleoproteins unperturbed. Altogether, we propose that phosphorylation of SRRM1/2 by TAOK2 plays a structural maintenance role that impacts SR protein-driven exon inclusion at the nuclear speckle.

molecular biology↗

bioRxiv2025

Class I histone deacetylases catalyze lysine lactylation

Metabolism and post-translational modifications (PTMs) are intrinsically linked and the number of identified metabolites that can covalently modify proteins continues to increase. This metabolism/PTM crosstalk is especially true for lactate, the product of anaerobic metabolism following glycolysis. Lactate forms an amide bond with the {varepsilon}-amino group of lysine, a modification known as lysine lactylation, or Kla. Multiple independent mechanisms have been proposed in the formation of Kla, including p300/CBP-dependent transfer from lactyl-CoA, via a high-energy intermediate lactoylglutathione species that non-enzymatically lactylates proteins, and several enzymes are reported to have lactyl transferase capability. We recently discovered that class I histone deacetylases (HDACs) 1, 2, and 3 can all reverse their canonical chemical reaction to catalyze lysine {beta}-hydroxybutyrylation. Here we tested the hypothesis that HDACs can also catalyze Kla formation. Using biochemical, pharmacological, and genetic approaches, we found that HDAC-catalyzed lysine lactylation accounts for the majority of Kla formation in cells. Dialysis experiments confirm this is a reversible reaction that depends on lactate concentration. We also directly quantified intracellular lactyl-CoA and found that Kla abundance can be uncoupled from lactyl-CoA levels. Therefore, we propose a model in which the majority of Kla is formed through enzymatic addition of lactate by HDACs 1, 2, and 3.

biochemistry↗

bioRxiv2024

A selective S-acyltransferase inhibitor suppresses tumor growth

S-acyltransferases play integral roles in essential physiological processes including regulation of oncogenic signaling pathways. While discovered over 40 years ago the field still lacks specific S-acylation inhibitors thus the potential benefit of pharmacologically targeting S-acyltransferases for human disease is still unknown. Here we report the identification of an orally bioavailable acyltransferase inhibitor SD-066-4 that inhibits the acyltransferase ZDHHC20. We identified a specific alanine residue that accommodates the methyl group of SD-066-4, thus providing isoform selectivity. SD-066-4 stably reduces EGFR S-acylation in Kras mutant cells and blocks the growth of Kras mutant lung tumors extending overall survival. We find that lung cancer patients harboring deletions in ZDHHC20 or ZDHHC14 concurrent with Kras alterations have a significant survival benefit, underscoring the translational importance of these enzymes.

cancer biology↗

bioRxiv2024

Intracellular Protein Editing to Enable Incorporation of Non-Canonical Residues into Endogenous Proteins

The ability to study proteins in a cellular context is crucial to our understanding of biology. Here, we report a new technology for "intracellular protein editing", drawing from intein- mediated protein splicing, genetic code expansion, and endogenous protein tagging. This protein editing approach enables us to rapidly and site specifically install residues and chemical handles into a protein of interest. We demonstrate the power of this protein editing platform to edit cellular proteins, inserting epitope peptides, protein-specific sequences, and non-canonical amino acids (ncAAs). Importantly, we employ an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented here will be applied to a diverse set of problems, enabling novel experiments in live mammalian cells and therefore provide unique biological insights. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/602493v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@8957b8org.highwire.dtl.DTLVardef@de02beorg.highwire.dtl.DTLVardef@1863cb4org.highwire.dtl.DTLVardef@1fb8bc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The Interplay of Acetylation and Ubiquitination Controls PRMT1 Homeostasis

PRMT1 plays many important roles in both normal and disease biology, thus understanding its regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.

biochemistry↗

bioRxiv2023

Reversible histone deacetylase activity catalyzes lysine acylation

Starvation and low carbohydrate diets lead to the accumulation of the ketone body, {beta}-hydroxybutyrate (BHB), whose blood concentrations increase more than 10-fold into the millimolar range. In addition to providing a carbon source, BHB accumulation triggers lysine {beta}-hydroxybutyrylation (Kbhb) of proteins via unknown mechanisms. As with other lysine acylation events, Kbhb marks can be removed by histone deacetylases (HDACs). Here, we report that class I HDACs unexpectedly catalyze protein lysine modification with {beta}-hydroxybutyrate (BHB). Mutational analyses of the HDAC2 active site reveal a shared reliance on key amino acids for classical deacetylation and non-canonical HDAC-catalyzed {beta}-hydroxybutyrylation. Also consistent with reverse HDAC activity, Kbhb formation is driven by mass action and substrate availability. This reverse HDAC activity is not limited to BHB but also extends to multiple short-chain fatty acids. The reversible activity of class I HDACs described here represents a novel mechanism of PTM deposition relevant to metabolically-sensitive proteome modifications.

molecular biology↗