Congestive heart failure (CHF) is characterized by the activation of neurohumoral drive concomitant with avid fluid retention. Renal denervation alleviates this fluid retention. SGLT2 (sodium-glucose cotransporter 2) inhibitors have shown remarkable improvement in patients with cardiovascular diseases. We have recently demonstrated a relationship between enhanced renal sympathetic nerve activity and SGLT2 expression as well as function during CHF; however, the precise molecular mechanisms involved in the expression and translocation of SGLT2 and associated scaffolding proteins to the luminal membrane remain to be examined. CHF was induced by coronary artery ligation followed by bilateral renal denervation 4 weeks later, in rats. Western blot analysis and immunohistochemistry were performed to evaluate changes in the expression of SGLT2, MAP17 (membrane-associated protein 17), PDZK1 (PDZ domain containing 1), and activation of ERK (extracellular signal-regulated kinase)/NF-KB (nuclear factor κB) in renal cortex. Human adult proximal tubular cells were used to determine the direct effect of norepinephrine on the expression of SGLT2-MAP17-PDZK1 and activation of the ERK/NF-KB pathway. Rats with CHF exhibited significantly enhanced expression of SGLT2, MAP17, and PDZK1 with a concomitant significant activation of ERK and NF-KB in the renal cortex. In rats with CHF, renal denervation mitigated enhanced expression of SGLT2-MAP17-PDZK1 as well as activation of ERK and NF-KB. Direct action of norepinephrine on human adult proximal tubular cells cells triggered enhanced expression of SGLT2-MAP17-PDZK1 by the activation of the ERK/NF-KB pathway. Enhanced basal renal sympathetic nerve activity in CHF activates the ERK/NF-KB pathway, which in turn facilitates the enhanced expression and translocation of the SGLT2-MAP17-PDZK1 scaffolding protein complex to the luminal membrane, augmenting sodium reabsorption in CHF.

Cytoplasmic aggregation of nuclear proteins such as TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) is associated with several neurodegenerative diseases. Studies in higher cells suggest that aggregates of TDP-43 and FUS sequester polysomes by binding RACK1 (receptor for activated C kinase 1), a ribosomal protein, thereby inhibiting global translation and contributing to toxicity. However, RACK1 is also a scaffold protein with a role in many other cellular processes including autophagy. Using yeast, we find that deletion of the RACK1 ortholog, ribosomal protein ASC1, reduces TDP-43 toxicity, but not FUS toxicity. TDP-43 foci remain liquid like in the absence of ASC1 but they become smaller. This is consistent with findings in mammalian cells. However, using double label fluorescent tags and co-immunoprecipitation we establish that ASC1 does not co-localize with TDP-43 foci, challenging the polysome sequestration hypothesis. Instead, ASC1 appears to influence toxicity through regulation of autophagy. We previously showed that TDP-43 expression inhibits autophagy and TOROID (TORC1 Organized in Inhibited Domains) formation and that genetic modifiers that rescue yeast from TDP-43 toxicity reverse these effects. Here we show that FUS does not inhibit autophagy. Deletion of ASC1 enhances a non-canonical form of autophagy that effectively counteracts TDP-43 induced autophagy inhibition despite reduced TOROID formation. Our findings highlight autophagy-not polysome sequestration-as a key mechanism underlying ASC1-mediated modulation of TDP-43 toxicity and suggest autophagy as a promising therapeutic target.

AHNAK Inhibits Osteoporosis Progression by Stabilizing Smad1 Protein.

Cells process signals by using large and complex networks of molecules that interact and modify one another. Some of these interactions occur among molecules connected by long flexible tethers, often made of intrinsically disordered protein regions. In this review, we present recent research showing that tethered reactions (a) are ubiquitous in cells, (b) are exploited by cell signaling networks, (c) can be qualitatively and quantitatively understood using simple polymer physics, (d) give rise to categorically different features compared with molecular interactions driven by free diffusion, and (e) provide novel avenues for therapeutics and bioengineering. Recent studies have begun to shed light on cases in which the tethers must reach between different molecular assemblies that are not connected by protein scaffolding. We provide an in-depth case study of immune receptors, where such tethered signaling plays a vital role in signal integration and immune cell decisions.

Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterised by remarkable cellular heterogeneity, which emerges early from the interplay of oncogenic KRAS signalling and inflammatory injury. However, the transcriptional, metabolic and functional properties of these pre-malignant cell states that initiate and drive PDAC progression remain elusive. This study aimed to identify and functionally characterise the critical premalignant cell states that arise from this heterogeneity, to define novel biomarkers and targets for early intervention. Public and in-house scRNA-seq data of pancreatic tumour models were analysed to identify key subpopulations in early cellular heterogeneity. Genetic perturbation in KrasG12D-driven models was performed to assess functional impact. Mechanistic studies used TurboID proximity proteomics, epigenetic profiling and metabolic assays. Clinical relevance was validated in human PDAC cohorts. We identified LY6D as a marker of a distinct, gastric-like cell state that emerges early and persists throughout tumourigenesis. The LY6D+ population exhibits conserved stemness and a unique, pan-stage dependency on oxidative phosphorylation (OXPHOS). Genetic ablation of Ly6d specifically impaired the gastric lineage and delayed tumourigenesis, while its overexpression enhanced tumourigenic and metastatic potential. Mechanistically, the glycosylphosphatidylinositol (GPI)-anchored LY6D protein scaffolds a lipid raft-associated kinase network that drives FOSL1-dependent epigenetic-transcriptional reprogramming. In human PDAC, LY6D+ cells harbour stemness and Epithelial-Mesenchymal Transition (EMT) signatures, and high LY6D expression is an independent prognostic marker of poor survival. Our work defines the LY6D+ gastric-like cell state as a key driver linking early pre-malignant heterogeneity to PDAC initiation and progression. LY6D represents a pan-stage therapeutic target and a candidate biomarker for early detection and therapeutic targeting.

As the diversity of therapeutic protein structures continues to evolve, it is essential to understand the mechanisms that determine their pharmacokinetic properties. The current work was initiated to establish the physicochemical attributes and cellular processes most crucial for the target-independent disposition of proteins possessing a fragment crystallizable (Fc) region. We systematically redesigned the surface properties of five de novo-generated protein scaffolds lacking any known binding partner in mice to produce a total of 35 Fc-fused proteins exhibiting a diverse set of physicochemical characteristics. Pharmacokinetic studies in wild-type mice revealed a profound spread in elimination rates and extensive tissue accumulation that was most strongly associated with charge descriptors. A suite of in vitro studies demonstrated that these in vivo observations significantly correlated to cellular nonspecificity wherein positive surface charge caused higher nonspecific adsorptive endocytosis, diminished recycling efficiency by the neonatal Fc receptor, and net cellular accumulation. Combined, our results provide a detailed explanation for how the disposition of Fc-fused proteins is impacted by charge, which will aid protein engineering efforts aimed at optimizing pharmacokinetic features.

Filoviruses, including Ebola (EBOV) and Marburg (MARV) viruses, are zoonotic pathogens that cause severe hemorrhagic fever in humans, with mortality rates reaching up to 90%. Filovirus egress and spread are driven by the viral matrix protein VP40 and regulated both positively and negatively by a growing number of specific host interactors. Here, we identify tetraspanin protein CD9, a plasma membrane organizing and scaffolding protein, as playing a role in facilitating efficient egress of EBOV and MARV. Indeed, we observed a significant decrease in viral egress of VLPs and live filoviruses from CD9-KD cells as compared to that from WT cells. Moreover, exogenous expression of CD9 rescued egress of VP40 VLPs close to WT levels in the CD9-KD cells. These findings identify tetraspanin CD9 as a positive regulator of filovirus egress, and thus CD9 may represent a potential new target for antiviral therapies targeting the late stage of the filovirus lifecycle.

Base excision repair (BER) is a critical pathway for repairing damaged DNA bases in cells; however, the mechanisms of protein recruitment and interaction in this pathway remain largely unexplored in higher plants. In this study, we used '84K' poplar (Populus alba × P. glandulosa) as the experimental system and applied a low concentration of 5-aminouracil (5-AU) to induce DNA base lesions. Through transcriptome analysis and weighted gene co-expression network analysis (WGCNA), we identified two key BER-responsive genes: the DNA glycosylase family gene PagDMG6341 and the DNA polymerase δ subunit PagPOLD4. PagDMG6341 was significantly upregulated during the arrest phase of 5-AU treatment, whereas PagPOLD4 expression peaked during the subsequent release phase. RNA interference (RNAi) lines for each gene resulted in impaired growth and increased susceptibility to 5-AU in '84K' poplar, supporting their functional roles in DNA repair and development. To further investigate their potential interaction network, we performed yeast two-hybrid (Y2H) screening, AlphaFold3-based structural modelling, confirmatory Y2H, bimolecular fluorescence complementation (BiFC) assays, and luciferase complementation imaging (LCI) assays. These experiments demonstrated that a Transducin/WD40-repeat-like scaffold protein (PagWD40) interacts independently with both PagDMG6341 and PagPOLD4. The yeast three-hybrid (Y3H) assay further showed that PagWD40 functions as a molecular scaffold, linking PagDMG6341 and PagPOLD4 to form a functional complex. This study reveals a new mechanism in which PagWD40 functions as a scaffold protein linking a DNA glycosylase with DNA polymerase δ in the plant BER pathway, thereby providing new insights into the organisation of plant DNA damage repair networks.

A novel and efficient method for the C(3)-H alkenylation of quinoxalin-2(1H)-ones has been developed using trifluoroacetic acid (TFA) as a Brønsted acid catalyst and Hantzsch esters (HEs) as the alkenylating agent. This metal-free protocol provides direct access to structurally diverse quinoxalinone-pyridine hybrid scaffolds under mild conditions, offering excellent functional group tolerance with yields ranging from good to high. The scope of the reaction was demonstrated by synthesizing 36 examples of quinoxalinone-pyridines in yields ranging from 61% to 82%. Quinoxalinone ring-containing drugs are well known for different pharmaceutical activities and, therefore, in the present case, we conducted a thorough in silico screening of the synthesized molecules (3a-3e') to elucidate their potent biological activities. We have adopted standard computational protocols, including DFT calculations, ADMET analysis, pharmacophore mapping, and molecular docking with proteins, to study the optimized geometries of the synthesized ligands. Protein scaffolds associated with cancer, diabetes, inflammation, and antimicrobial activity were targeted to investigate the drug likeness. The method revealed that compounds 3h-3m, among the 31 molecules, show high potential as viable drugs. Specifically, 3l yielded the best docking result, and the MD simulation indicated that 3l has potential as a drug candidate.

Dendritic spine morphology is strongly associated with neurodevelopmental disorders. Synaptic plasticity alters spine volume, a phenomenon known as structural plasticity, which influences information processing within neuronal circuits. Structural changes at dendritic spines are linked to autism spectrum disorders (ASD), particularly those involving gene mutations that result in synaptopathy. Loss of a single copy of the Shank3 gene leads to Phelan-McDermid syndrome, a synaptopathy, as Shank3 encodes SHANK3, a scaffold protein in the postsynaptic density of glutamatergic neurons. In this study, the structural plasticity of dendritic spines was evaluated in male and female Shank3+/- and wild-type (WT) mice in response to synaptic plasticity. Two-photon imaging and glutamate uncaging were employed in organotypic hippocampal cultures. Cognitive function in adult Shank3+/- mice was also assessed using a novel object recognition test. The results indicate that Shank3+/- mice exhibit altered structural plasticity in response to long-term depression (LTD) and display a heterosynaptic response in neighboring spines. Increased GluN2B expression and NMDA currents underlie these effects and may influence object recognition memory in Shank3+/- mice. These findings suggest that Shank3 haploinsufficiency induces synaptic alterations during postnatal development that impact memory in adulthood.Significance statement Alterations in the morphology and density of dendritic spines, which are the sites of approximately 90% of synapses, have been associated with neurodevelopmental disorders such as autism spectrum disorder (ASD). Although these associations have been established, it is unclear how dendritic spine structural changes affect synaptic responses and cognitive function in ASD. This study uses a genetic mouse model of ASD (Shank3 heterozygous mice with a deletion of exons 4-9) to investigate synaptic responses and their structural correlates. The aim is to elucidate the neurobiology of ASD and determine how these modifications may affect memory in adulthood.

Lung adenocarcinoma (LUAD) remains a major global health issue characterized by high incidence and mortality rates. RecQ-like helicase 4 (RECQL4), a member of the DNA helicase family, plays a crucial role in DNA replication, DNA damage repair, and tumor progression. However, its involvement and specific molecular mechanisms in LUAD progression have not been elucidated. Through this investigation, we found that RECQL4 expression was aberrantly elevated in clinical LUAD tissues, and higher levels of RECQL4 expression were associated with poor prognosis and worse clinicopathological characteristics in LUAD patients. Gain-of-function and loss-of-function studies demonstrated that RECQL4 promoted the proliferation, migration, and invasion abilities of LUAD cells. Subsequent gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis confirmed that RECQL4 activates the NF-κB signaling pathway. Mechanistic investigation indicated that RECQL4 might function as a scaffold protein for the Y box binding protein 1 (YBX1) and GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), enhancing the interaction between YBX1 and G3BP1, thereby activating the NF-κB signaling pathway and promoting the progression of LUAD. In conclusion, RECQL4 promotes the malignant progression of LUAD through the YBX1/G3BP1-mediated NF-κB signaling pathway. These findings suggest that RECQL4 has the potential to serve as a novel prognostic biomarker and an effective therapeutic target for LUAD.

Meet the author: Cathleen Zeymer.

Viral myocarditis is a major cause of sudden cardiac death and can lead to dilated cardiomyopathy in adults. However, effective treatments remain elusive due to an incomplete understanding of its molecular drivers. Here, we investigate the role of β-arrestins (βarrs), scaffolding proteins that regulate GPCR signaling, in acute viral myocarditis. Using global βarr1 and βarr2 knockout (KO) mice, we assessed immune cell infiltration and apoptosis as markers of cardiac inflammation under Coxsackievirus (CVB3) infection. CVB3-infected βarr1 and 2 KO mice exhibited suppressed recruitment of NK cells, monocytes, macrophages, dendritic cells, and T cells over a broad range of viral titers at 7 days post-infection along with reduced cardiac apoptosis. At 4 days post-infection, immune cell expansion in secondary lymphoid organs, including B cells, CD8+ T cells, CD64+ myeloid progenitors, and monocyte/macrophages was also impaired in βarr KO mice. Importantly, cardiomyocyte-specific βarr1 and 2 dual deletion mirrored the attenuated inflammatory response and apoptosis observed in global βarr KO mice. Mechanistically, cardiomyocytes lacking βarr1 or βarr2 displayed defective cGAS-STING pathway activation, with impaired STING, TBK1, and IRF3 phosphorylation and inhibited IFNβ production at 24 hours post-CVB3 infection. These data highlight βarrs as critical mediators of the inflammatory response in the heart and secondary lymphoid organs during viral myocarditis and demonstrate that cardiomyocyte βarrs play a fundamental role in the inflammatory response to CVB3 viral myocarditis.

Identification of FocAntigen1 as a pathogenicity effector in Fusarium oxysporum f. sp. cubense tropical race 4 targeting the scaffold protein MaRACK1 in banana

The oxygen sensitivity of [4Fe-4S] clusters on the nitrogenase scaffold protein NifU.

Human genetics guides the discovery of CARD9 inhibitors with anti-inflammatory activity.

Rationale: The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (KSR2) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms.Methods: Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of KSR2 in atherosclerosis, we utilized global KSR2 knockout mice fed a high-fat diet ad libitum, pair-fed global KSR2 and Apoe (Apolipoprotein E) double knockout mice, and mice with endothelial-specific KSR2 overexpression mediated by AAV9-ICAM2.Results: Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of KSR2 expression via differential binding of the transcriptional repressor XBP1s. KSR2 expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple KSR2 gene-edited mouse models, we demonstrated that endothelial KSR2 protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4ACRBN E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1. The subsequently activated AMPK signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects.Conclusions: Our findings provide the first comprehensive molecular explanation of the rs12822146-KSR2-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.

Tip growth is closely tied to fungal pathogenicity. Budding yeast Spa2 (the homolog of GIT1 and GIT2 in mammals), a multi-domain protein and member of the polarisome, orchestrates tip growth in yeasts and other fungi. We identified a conserved short linear motif in the Rab GTPase-activating proteins (RabGAPs) Msb3 and Msb4, and the MAP kinase kinases Ste7 and Mkk1, which mediates their interaction with Spa2. AlphaFold predictions suggest that these initially unstructured motifs adopt an α-helical conformation upon binding to the hydrophobic cleft in the N-terminal domain of Spa2. Altering the predicted key contact residues in either Spa2 or the motif reduces complex stability. Such mutations also cause mis-localization of Msb3, Msb4 and Ste7 within the cell. Deleting the motif in Msb3 or Msb4 abolishes tip-directed growth of the yeast bud. Protein assemblies that spatially confine secretion to specific membrane regions are a common feature of eukaryotic cells. Accordingly, complexes between proteins with this motif and Spa2 were predicted in orthologs and paralogs across selected Opisthokonta, including pathogenic fungi and humans. A search for functional motifs in conformationally flexible regions of all yeast proteins identified Dse3 as a novel Spa2-binding partner.

Children with autism often exhibit abnormalities in body weight, but the underlying mechanism remains unclear. SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a scaffold protein of the postsynaptic density, has been reported to be associated with autism. This study aimed to investigate whether and how SHANK3 influences body weight in the hypothalamic neuronal regulation of energy homeostasis. Adeno-associated viruses 9 (AAV9) carrying CMV-Cre and Agrp-Cre were stereotactically injected to restore SHANK3 expression in the arcuate nucleus (ARC) and agouti-related peptide (AgRP) neurons, respectively. Agrp-Cre mice were injected with AAV9-p38αflox/flox to overexpress p38α. Activated p38α was generated by mutating both D176A and F327S in p38α. Inactivated p38α was constructed by mutating both T180A and Y182F in p38α. Metabolic analysis, immunoblotting, histological analysis, the glucose tolerance test, the insulin tolerance test, and body fat mass analysis were applied to investigate the underlying mechanisms by which SHANK3 regulates body weight. We reveal that SHANK3 regulates body weight via the p38α signaling pathway in the AgRP neurons of the hypothalamus. Shank3 knockout (Shank3-/-) mice exhibit resistance to diet-induced obesity. Shank3 re-expression in the ARC or AgRP neurons increases body weight in Shank3 knock-in mice with an inverted allele (SKO). Overexpression or activation of p38α in AgRP neurons elicits resistance to diet-induced obesity. Inactivated p38α in AgRP neurons abolished the resistance to diet-induced obesity due to SHANK3 deficiency. Our findings suggest that the SHANK3-p38α siganling pathway in AgRP neurons regulates body weight balance in autism, revealing a promising therapeutic target for obesity in children with autism.