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Effect of a Laparoscopic Donor Nephrectomy in Healthy Living Kidney Donors on the Acute Phase Response Using Either Propofol or Sevoflurane Anesthesia
Surgical trauma elicits a complex inflammatory stress response, contributing to postoperative morbidity and recovery variability. This response is influenced by patient-specific factors and surgical and anesthetic techniques. To isolate the impact of anesthesia on the acute phase response, we investigated plasma proteomic changes in a uniquely homogeneous cohort of healthy, living kidney donors (n = 36; propofol = 19; sevoflurane = 17) undergoing laparoscopic donor nephrectomy. Proteomic profiling of plasma samples collected preoperatively and at 2 and 24 h postoperatively revealed 633 quantifiable proteins, of which 22 showed significant perioperative expression changes. Eight proteins exhibited over two-fold increases, primarily related to the acute phase response (CRP, SAA1, SAA2, LBP), tissue repair (FGL1, A2GL), and anti-inflammatory regulation (AACT). These changes were largely independent of anesthetic type, though SAA2 and MAN1A1 showed anesthetic-specific expression. The upregulation of these proteins implicates the activation of immune pathways involved in host defense, tissue remodeling, and inflammation resolution. Our findings provide a molecular reference for the surgical stress response in healthy individuals and highlight candidate biomarkers for predicting and managing postoperative outcomes. Understanding these pathways may support the development of strategies to mitigate surgical stress and enhance recovery, particularly in vulnerable patient populations.
N-terminal cysteine acetylation and oxidation patterns may define protein stability
AbstractOxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.
Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.
Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
Drug Combinations Targeting FAK and MEK Overcomes Tumor Heterogeneity in Glioblastoma
Background/Objectives: Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor prognosis, largely owing to its heterogeneity and the involvement of multiple intracellular signaling pathways that contribute to drug resistance. While recent advancements in targeted drug combination therapies, such as dabrafenib and trametinib, show promise for certain GBM subgroups, identifying effective drug combinations across the broader GBM population remains a challenge. Integrin-mediated signaling, particularly through Focal Adhesion Kinase (FAK), plays a pivotal role in GBM pathogenesis and invasion, making it a potential therapeutic target and component of future drug combination strategies. Methods: In this study, we utilized a chemogenomic screening approach to identify synergistic drug combinations that target FAK in glioblastoma. We initially employed a CRISPR-engineered GBM model to assess the effects of FAK depletion and subsequently discovered that combining FAK inhibitors such as VS4718 with MEK inhibitors, particularly trametinib, demonstrated synergistic effects. This potent combination was validated using various 2D and 3D assays, including cell viability/apoptosis assessment, synergistic analysis, cellular imaging, and target engagement assays. This combination also effectively inhibited spheroid growth and invasion across a diverse panel of patient-derived GBM stem cells. Molecular mechanisms underlying these effects include suppression of multiple kinase signaling pathways and enhanced apoptosis, elucidated using Reverse-Phase Protein Array (RPPA) profiling and Western blot validation. Result: In vivo, combination therapy significantly reduced the tumor volume in orthotopic transplantation models. Conclusions: These findings suggest that the combination of FAK and MEK inhibitors represents a promising therapeutic strategy to overcome the challenges of GBM treatment.
Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium
The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum survival and represents a potential target for antimalarial therapies. We utilised a ubiquitin- activity based probe (Ub-Dha) to capture active components of the ubiquitin conjugating machinery during asexual blood-stage development. Several E2 ubiquitin-conjugating enzymes, the E1 activating enzyme, and the HECT E3 ligase PfHEUL were identified and validated through in vitro ubiquitination assays. We also demonstrate selective functional interactions between PfHEUL and a subset of both human and P. falciparum E2s. Additionally, the Ub-Dha probe captured an uncharacterized protein, PF3D7_0811400 (C0H4U0) with no known homology to ubiquitin-pathway enzymes in other organisms. Through structural and biochemical analysis, we validate it as a novel E2 enzyme, capable of binding ubiquitin in a cysteine-specific manner. These findings contribute to our understanding of the P. falciparum UPS, identifying promising novel drug targets and highlighting the evolutionary uniqueness of the Ub-proteasome system in this parasite.
Allosteric communications between domains of nuclear receptors.
Nuclear receptors (NRs) regulate gene expression in response to hormonal signals, influencing diverse physiological processes and diseases. Structural and dynamics investigations based on X-ray crystallography, cryo-electron microscopy (cryo-EM), hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulations, have significantly deepened our understanding of the conformational states, dynamics, and interdomain interactions of multi-domain NRs. Structural studies have examined heterodimeric complexes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) with retinoid X receptor alpha (RXRα), liver X receptor beta (LXRβ) with RXRα, and retinoic acid receptor beta (RARβ) with RXRα, as well as homodimers like hepatic nuclear factor 4 alpha (HNF-4α), androgen receptor (AR), and glucocorticoid receptor (GR). These investigations highlight critical allosteric communication between ligand-binding domains (LBDs) and DNA-binding domains (DBDs), emphasizing the roles of flexible hinge regions and N-terminal segments in adapting to diverse DNA configurations. Both non-steroid receptor heterodimers and homodimers exhibit robust interdomain connections that mediate allosteric signaling. For instance, AR demonstrates three distinct conformational states that underscore its dynamic behavior, while GR exhibits unique ligand-dependent domain interactions shaping receptor signaling. The collective findings so far suggest a conserved mechanism of cross-domain communication across the NR family. Supported by complementary biophysical, spectroscopic, mutagenesis, and computational studies, this body of research has elucidated the nature of domain-domain interfaces and their pivotal roles in regulating the transcriptional activity of steroid and non-steroid receptors.
Single-nucleus multi-omics implicates androgen receptor signaling in cardiomyocytes and NR4A1 regulation in fibroblasts during atrial fibrillation
Abstract The dysregulation of gene expression programs in the human atria during persistent atrial fibrillation (AF) is not completely understood. Here, we reanalyze bulk RNA-sequencing datasets from two studies (N = 242) and identified 755 differentially expressed genes in left atrial appendages of individuals with persistent AF and non-AF controls. We combined the bulk RNA-sequencing differentially expressed genes with a left atrial appendage single-nucleus multi-omics dataset to assign genes to specific atrial cell types. We found noncoding genes at the IFNG locus (LINC01479, IFNG-AS1) strongly dysregulated in cardiomyocytes. We defined a gene expression signature potentially driven by androgen receptor signaling in cardiomyocytes from individuals with AF. Cell-type-specific gene expression modules suggested an increase in T cell and a decrease in adipocyte and neuronal cell gene expression in AF. Lastly, we showed that reducing NR4A1 expression, a marker of a poorly characterized human atrial fibroblast subtype, fibroblast activation markers, extracellular matrix remodeling and cell proliferation decreased.
Pharmacological targeting of BMAL1 modulates circadian and immune pathways.
The basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) proteins BMAL1 and CLOCK heterodimerize to form the master transcription factor governing rhythmic gene expression. Owing to connections between circadian regulation and numerous physiological pathways, targeting the BMAL1-CLOCK complex pharmacologically is an attractive entry point for intervening in circadian-related processes. In this study, we developed a small molecule, Core Circadian Modulator (CCM), that targets the cavity in the PASB domain of BMAL1, causing it to expand, leading to conformational changes in the PASB domain and altering the functions of BMAL1 as a transcription factor. Biochemical, structural and cellular investigations validate the high level of selectivity of CCM in engaging BMAL1, enabling direct access to BMAL1-CLOCK cellular activities. CCM induces dose-dependent alterations in PER2-Luc oscillations and orchestrates the downregulation of inflammatory and phagocytic pathways in macrophages. These findings collectively reveal that the BMAL1 protein architecture is inherently configured to enable the binding of chemical ligands for functional modulation.