Early-stage drug discovery relies on the availability of stable protein for reliable biophysical characterization of ligand binding. However, many Plasmodium falciparum proteins are challenging to produce in heterologous systems, which limits their experimental utility. To address this, we tested whether ProteinMPNN-guided sequence design could generate stabilized surrogate constructs that retain wild-type-like structure and binding thermodynamics. Designs were generated with constraints to maintain conserved and binding-site residues for three therapeutically relevant targets: PfBDP1-BRD, PfBDP4-BRD, and PfK13-KREP. The resulting constructs showed markedly increased thermal stability. Using PfBDP1-BRD as a benchmark, isothermal titration calorimetry confirmed that the stabilized variants retained wild-type-like binding thermodynamics with a known ligand. Extending this approach to other targets, a PfK13-KREP construct led to an apo structure with a binding pocket closely matching the wild type. For PfBDP4-BRD, virtual screening against a previously reported wild-type crystal structure identified putative binders, while a stabilized surrogate for this otherwise unstable target enabled their experimental validation and the determination of a 1.25 Å co-crystal structure with a newly identified inhibitor. Our findings demonstrate that computationally stabilized surrogates are practical and effective tools for robust biophysics and structure-enabled drug discovery against otherwise challenging malaria proteins.
Irene Schulz passed away on Sunday 29th March 2026. She made fundamental contributions to the field of Calcium Signaling and, in collaboration with the late Sir Michael Berridge, showed directly that inositol-1,4,5 trisphosphate (IP3) released Ca2+ from a non-mitochondrial intracellular store that she later identified as the endoplasmic reticulum (ER). The discovery of IP3 as a Ca2+ releasing messenger was a momentous event in the field of signal transduction mechanisms and was foundational for the field of Ca2+ Signaling. Irene was born in Berlin on 25th January 1941 and graduated from the Marie-Curie High School in Berlin in February 1960. She undertook her medical studies at the Free University of Berlin and graduated in Medicine in 1966. She then went to the US, as a Postdoctoral Fellow at the National Institutes of Health (NIH) in Bethesda. In 1967, she returned to Europe to become Group Leader at the Max-Planck-Institute for Biophysics in Frankfurt, under the directorship of Karl Ullrich. From 1976 - 1991 she was also an Associate Professor of Physiology at the JW Goethe University in Frankfurt. In 1991, Irene became a full Professor and Director of the 2nd Physiological Institute at the University of the Saarland, Homburg/Saar and was in charge of that Institute until her retirement in 2006.
Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on epithelial cell shape and actin dynamics. 12Z human endometriotic epithelial cells were transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 h, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-h E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 min of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.
Sepiapterin reductase (SPR) catalyzes several key steps in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases, and therefore for neurotransmitter production. Although several reductases-including carbonyl reductase 1 (CBR1), aldose reductase (AKR1B1), and AKR1C3-can substitute for SPR activity in vitro, their physiological significance remains unresolved. This study examines AKR1C3 as a component of an alternative BH4-generating pathway and evaluates its capacity to compensate for BH4 loss under diminished SPR activity. In vitro assays identified 2'-OXPH4 as the primary product of AKR1C3, redirecting pathway flux away from the canonical 1'-OXPH4 intermediate and the sepiapterin-salvage pathway. To assess the occurrence and efficiency of this route in cells, we generated SPR-knockout (SPR-KO) cell and evaluated pathway products in SPR-KO and wild-type (WT) backgrounds. In WT cells neither AKR1C3 nor SPR overexpression altered BH4 synthesis, indicating that neither enzyme is rate-limiting. In contrast, AKR1C3 increased BH4 levels in SPR-KO cells, while inhibiting sepiapterin production, revealing that AKR1C3 becomes functionally engaged only when SPR activity is decreased. Based on relative enzyme abundance, AKR1C3 and SPR exhibited comparable catalytic efficiency in this context. Importantly, AKR1C3-mediated BH4 production was sufficient to sustain tyrosine hydroxylase (TH) activity in SPR-KO cells, as demonstrated by L-DOPA formation. These findings establish AKR1C3-driven 2'-OXPH4 synthesis as a bona fide, inducible pathway capable of maintaining BH4 levels when SPR activity is limiting. This alternative path provides a compelling therapeutic target and introduces a new diagnostic consideration for patients with diminished SPR activity.
Chlamydomonas reinhardtii is an ideal model organism for studying the cytoplasmic preassembly of ciliary dyneins. However, under normal liquid-culture conditions, Chlamydomonas preassembly-deficient mutants often show no cilia or a small number of ciliated cells (i.e., low ciliation ratio), which hinders further analysis of both ciliary dyneins and the phenotypes of these mutants. In this brief report, we present a modified culture method for one of the Chlamydomonas preassembly-deficient mutants, pf23 . This method enables researchers to obtain enough pf23 cilia for small-scale biochemical, biophysical, structural and phenotypic analyses.
Patients with Down Syndrome (DS) are characterized by dysfunction of several organs, including the liver, brain, heart defects, gastrointestinal anomalies, and lethal immune hypersensitivity. A person with DS is also susceptible to various inflammatory diseases, including hepatic autoimmune diseases. The Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is known to trigger the stimulator of interferon genes (STING) and downstream proinflammatory factors. In this work, we hypothesized that oxidative stress-associated DNA damage triggers activation of the cGAS-STING signaling pathway and promotes liver inflammation in DS. Here, we investigated the role of reactive oxygen species (ROS) associated DNA damage and the cGAS-STING signaling pathway in the pathogenesis of hepatic inflammation in the DS model. Our results showed that DS cells harbor excessive ROS and DNA damage in DS fibroblasts and DS mouse liver. Further, DS cells accumulate micronuclei that likely serve as a source of cytoplasmic DNA to stimulate cGAS-STING activation. In addition, RNA-seq analysis results showed enhanced expression of key type I interferon factors in cGAS-STING pathways in DS liver and inflammatory responses and elevated liver enzymes such as alanine transaminase (ALT) that indicate a hepatocellular liver injury in DS. The results of this study opened the opportunity to connect endogenous DNA damage triggers innate immune response, which may contribute to the upregulation of the cGAS-STING signaling to exacerbate hepatic inflammation in DS.
Hydrostatic pressure in living organisms is crucial for the formation and stability of hollow structures in tissues and organs. However, the underlying mechanisms governing the collective cell responses to pressure in these processes have not yet been fully understood. Here, we developed a hydrostatic pressure generator to produce various pressures of physiological magnitudes and explored their effects on dome structure formation in the epithelial monolayer. We found that the positive hydrostatic pressure promoted dome formation, while the negative one suppressed it. The positive pressure induced cell autophagy and thus increased transepithelial electrical resistance, which elevated osmotic pressures inside the dome. In addition, the positive pressure induced reorganization of the actin-cytoskeleton, which stabilized the cytoskeleton network and weakened cell-matrix adhesion. Interestingly, during dome expansion, the negative pressure promoted the expansion, which eventually led to dome rupture, while the positive pressure suppressed the expansion and subsequent rupture. Our numerical simulations revealed that the negative pressure produced larger intercellular normal stress within the dome wall, making the dome more prone to rupture. These findings revealed the biophysical mechanisms by which hydrostatic pressure regulates dome formation and stability and provided insights into the effect of external pressure on collective cell behaviors during tissue morphogenesis.
To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics and the structural distributions they generate remains a key challenge. Here, using 1146 crystal structures of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the enzyme's structural fluctuations. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the structural changes that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally correlated with the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography, in which numerous crystallographic datasets are analyzed, can reveal the structural fluctuations of protein native states and help uncover their biological function.
Kv3.3 voltage-gated K+ (Kv) channels are highly expressed in cerebellar Purkinje neurons and some hippocampal neurons, aligning with the motor and cognitive impairments observed in spinocerebellar ataxia 13 (SCA13) caused by Kv3.3 mutations. Despite their functional significance, the mechanisms governing Kv3.3 subcellular localization remain poorly understood. Here we report microtubule-associated protein 6 (MAP6) regulates Kv3.3 axon-dendrite targeting. MAP6 deletion reduces Kv3.3 levels in the processes of Purkinje neurons. Mechanistically, MAP6's 1st and 2nd Mn modules directly bind the external surface of the Kv3.3 N-terminal T1 tetramer, while its 3rd Mn module indirectly associates with Cav2 Ca2+ channels. In Purkinje neurons, shRNA-mediated MAP6 knockdown decreases somatodendritic levels of both Kv3.3 and Cav2.1 (associated with SCA6). Notably, expression of Mn1/2-GFP selectively reduces Kv3.3, but not Cav2.1, levels. Purkinje neuron burst firing is reduced in both conditions. These findings uncover a MAP6-dependent mechanism for targeting two key ion channels linked to SCAs.
Gut microbiota and bile acids have been reported to affect sepsis progression, but the underlying mechanisms remain largely unknown. Here we investigated gut microbiota-bile acid interplay in two paediatric sepsis cohorts. Integration of bile acid-targeted metabolomics with gut metagenome data from paediatric sepsis patients identified deoxycholic acid 3-sulfate (DCA-3S) as significantly associated with paediatric sepsis progression. In vitro and in vivo experiments identified Enterococcus raffinosus as the primary producer of DCA-3S, contributing at least 80% of its total production, challenging the conventional notion of hepato-centric bile acid sulfation pathways. Intervention experiments in mouse and intestinal organoid models revealed that DCA-3S administration effectively alleviated sepsis by improving intestinal barrier function and attenuating inflammatory response. Collectively, our findings highlight a previously unrecognized microbial contribution to bile acid sulfation and position DCA-3S as a promising diagnostic and therapeutic biomarker for paediatric sepsis.
Coffee extracts contain numerous bioactive compounds. Given the dietary link between coffee consumption and colorectal cancer, this study compared the effects of roasted and green (unroasted) coffee extracts on human colorectal cancer cells (HCT116) and non-cancerous fibroblasts (BJ-5ta) to evaluate how processing influences proliferation and molecular signaling. Real-time cell analysis (RTCA), qRT-PCR, and label-free quantitative proteomic analysis were performed to assess cellular responses. MTS and RTCA showed that in BJ-5Ta fibroblasts, coffee extracts increased proliferation in the order CNR < CAR < CAU < CNU, whereas the trend was reversed in HCT116 cancer cells. Proteomic analysis revealed that in BJ-5Ta cells, unroasted coffee extract caused downregulation of the ribosome pathway, and natural coffee extract caused downregulation of the gap junction pathway, indicating reduced protein synthesis and cell-cell communication as a potential stress-adaptive response. In contrast, in HCT116 cells, unroasted coffee extract upregulated the ribosome pathway. Also, natural coffee extract upregulated the pentose phosphate pathway in HCT116 cells, which may enhance NADPH production and reduce oxidative stress. Current evidence suggests coffee's bioactive compounds may have different effects varying by coffee extract type and their preparation.
In the MA.32 randomized adjuvant breast cancer (bc) trial, metformin (vs placebo) did not impact invasive disease free (IDFS) or overall survival (OS) in estrogen/progesterone receptor (ER/PgR) positive or negative bc; exploratory analyses suggested a benefit in HER2 positive bc. We investigated whether body mass index (BMI) and obesity-associated blood variables predicted metformin benefit in immunohistochemically defined bc subtypes [luminal (ER/PgR positive, HER2 negative), triple negative (TNBC; ER, PgR, HER2 negative), HER2 positive]. 3649 non-diabetic patients with high risk T1-3, N0-3 M0 bc were randomized. Baseline fasting plasma was assayed for insulin, glucose, leptin, hsCRP; Homeostasis Model Assessment (HOMA) was calculated. For each bc subtype and each outcome [distant recurrence free survival (DRFS), OS, IDFS], Cox models examined interactions of Body Mass Index (BMI) and blood variables with metformin vs placebo outcomes. Mean age was 51.1 to 53.0 years; mean BMI 27.3 to 27.5 kg/m. 2 Most cancers were T2, N0 or N1 and grade 2 to 3; 2104 (57.7%) were luminal, 925 (25.3%) TN and 620 (17.0%) HER2 positive. Median follow-up 95.9 months. In luminal bc, significant interactions were identified for leptin, insulin and HOMA on DRFS and in TNBC a significant interaction was identified for glucose on DRFS, with potential adverse effects of metformin at lower levels of each variable. In those with HER2 positive bc, no variable predicted metformin benefit. BMI and blood variables did not identify subgroups with luminal or triple negative bc who benefitted from metformin nor those with HER2 positive bc who did not benefit.
This study aims to explore the anti-inflammatory mechanism of Anwulignan (AN) by integrating proteomics, molecular docking, and in vitro cell models. A lipopolysaccharide (LPS)-induced inflammatory model in RAW264.7 cells was established. The cells were divided into a control group, a model group (LPS treatment), and a drug treatment group (LPS + AN). Data-independent acquisition proteomics was employed to screen differentially expressed proteins using the thresholds of fold change greater than 1.2 or less than 0.8 and p < 0.05. Bioinformatics analysis and molecular docking were combined to identify core targets and regulatory pathways, which were subsequently validated by Western blot. A total of 129 potential anti-inflammatory targets and six core targets of AN were identified. KEGG enrichment analysis indicated that the anti-inflammatory effects of AN primarily involve protein processing in the endoplasmic reticulum, as well as the p53, FoxO, and RIG-I-like receptor signaling pathways. Molecular docking analysis revealed that AN exhibits strong binding affinities with these core targets. Western blot validation further confirmed that AN significantly downregulates the expression of pro-inflammatory proteins (CYCS, MAPK14, ATM, and EIF2AK2) and upregulates the expression of anti-inflammatory proteins (CDKN1A and RBX1) in RAW264.7 cells. AN exerts synergistic anti-inflammatory effects through a multi-target and multi-pathway manner, primarily by modulating core targets, along with their associated signaling pathways.
Cyclophosphamide (CYC) is a well-established nephrotoxic agent widely used in the treatment of cancer and various immunosuppressive disorders. Diosmin, a bioactive flavonoid, has been reported to reduce drug-induced adverse effects. This study evaluates the effects of diosmin on CYC-induced nephrotoxicity and explores potential mechanisms involved in CYC-related renal damage. Thirty-two male rats were randomly assigned to four experimental groups: control, diosmin, CYC, and CYC + diosmin. Diosmin (100 mg/kg) was administered once daily for 15 consecutive days, while cyclophosphamide (200 mg/kg) was given as a single dose on the 8th day of the experimental period. Diosmin was associated with attenuation of CYC-induced pathological alterations in renal tissue architecture. It reduced the immunoreactivity of pro-inflammatory mediators (IL-1β, IL-6, TNF-α, and iNOS) and modulated apoptosis by decreasing the pro-apoptotic marker BAX and increasing the anti-apoptotic marker Bcl-2. Although CYC treatment suppressed mTOR and increased SIRT1 immunoreactivity, diosmin co-administration attenuated these alterations. Additionally, markers of renal DNA damage were reduced. Overall, the findings suggest that diosmin may exert favorable effects in CYC-induced nephrotoxicity in this experimental model.
Histologically normal mammary tissue from breast cancer patients can harbor significant genetic alterations that could precede visible tumor development and influence disease progression. Whole-exome sequencing was performed on 408 samples from 77 breast cancer patients with poor prognosis, 49 patients recruited without prognosis-based selection, and 15 individuals undergoing non-cancer-related mammoplasty. Paired primary tumor and histologically normal mammary gland tissues were analyzed. Variant classification adhered to strict filtering criteria, incorporating allele frequency thresholds, multiple annotation databases, and in silico prediction tools. Duplex sequencing was employed to detect and confirm pathogenic PIK3CA and TP53 variants in normal mammary tissue samples from 11 breast cancer patients with unfavorable prognosis. Statistical analyses included hypergeometric testing, Kaplan-Meier survival analysis, and Cox proportional hazards modeling. Post-zygotic pathogenic variants in cancer-associated genes were significantly more prevalent in normal mammary tissue of poor-prognosis patients (29%) than in unselected patients (12.5%) (p = 0.0008578). Variant presence and per-individual burden were similar across age-matched cohorts and intrinsic subtypes, indicating that subtype composition, germline predisposition and age do not account for the excess UM variant load in BCAP. Truncating variants were exclusive to poor-prognosis cases. Frequently altered genes included AKT1, PIK3CA, PTEN, TBX3, and TP53, with TP53 variants detected only in patients with adverse outcomes. Duplex sequencing confirmed the presence of low-frequency variants (as low as 1.34%) in regions of histologically normal breast tissue from patients with a poor prognosis. Notably, nearly one-quarter of all identified cases (24%, 12/49) harbored pathogenic variants in normal tissue absent from corresponding primary tumors, suggesting that at least some mosaic clones in uninvolved mammary tissue represent independent evolutionary events rather than residual tumor cells. Post-zygotic pathogenic variants are frequent in histologically normal mammary tissue from breast cancer patients, including alterations in key cancer-associated genes. These findings indicate that mosaic clonal changes outside the tumor are more common than previously appreciated and warrant further investigation. Assessing such variants in non-tumorous tissue may, in the future, help refine approaches to breast cancer risk evaluation and management.
The LIM kinases (LIMK1/2) are key mediators in signaling cascades that regulate actin cytoskeleton dynamics via cofilin phosphorylation. Dysregulation of these pathways and overexpression of LIMKs are implicated in disease development, including cancer, Fragile X syndrome, and glaucoma. Positioned downstream of actin-regulating Rho GTPase signaling pathways, LIM kinases are attractive drug targets. Here, we targeted LIMKs with PROTACs to disrupt both catalytically and noncatalytically mediated functions. Despite employing a dual LIMK1/2 inhibitor warhead and high structural conservation between the two human LIM kinases, we discovered isoform-specific LIMK2 degradation by initial PROTACs that we optimized into a highly potent and selective LIMK2 degrader. Cell-based assays and structural analysis indicated that isoform specificity was likely driven by favorable orientation bias and/or lysine accessibility, along with enhanced ternary complex formation. We comprehensively characterized the PROTAC as a chemical probe that induces isoform-specific degradation, offering a powerful alternative to conventional reversible pan-LIMK inhibitors.
Autosomal dominant acute porphyrias are rare inherited disorders of haem biosynthesis characterised by accumulation of potentially neurotoxic porphyrin precursors and attacks of severe abdominal pain with autonomic and neuropsychiatric features. Disease severity ranges from asymptomatic individuals to those with recurrent, life-threatening attacks. The International Porphyria Network invited 34 acute porphyria specialists from 17 countries to form an expert panel. The invited group included clinicians from diverse specialities (ie, internal medicine, haematology, endocrinology, gastroenterology, hepatology, neurology, and biochemistry), together with laboratory scientists and patient representatives. The panel met online (in 2023-25) to develop 15 evidence-based recommendations with the use of the Grading of Recommendations, Assessment, Development, and Evaluations framework addressing attack prevention, management of sporadic and recurrent attacks, long-term follow-up, surveillance for primary liver cancer, and family screening. The guidelines support safe, consistent clinical care and improved outcomes, recognising global variation in resources and access to high-cost drugs, and highlighting priorities for future research.
Carbohydrates play essential roles in biological, atmospheric, and food-related systems, where their hydration characteristics regulate stability, reactivity, and macroscopic behavior. In this work, ATR-FTIR difference spectroscopy combined with spectral deconvolution was employed to elucidate how arabinose, galactose, and fructose modulate the hydrogen-bond network of water. Analysis of the integrated peak areas shows that sugar addition perturbs the ordered water structure and redistributes the populations of different hydrogen-bonded environments within the OH-stretch region, with fructose inducing the highest effect, followed by galactose and arabinose. As solute concentration increases, the progressive depletion of available water molecules enhances sugar-sugar interactions, further shaping the overall hydrogen-bonding landscape in bulk water. Moreover, fructose forms stronger hydrogen bonds with water relative to arabinose and galactose. These findings indicate that the hydration behavior of carbohydrates is governed predominantly by the number and spatial arrangement of hydroxyl groups rather than by the carbohydrate backbone, while the present approach enables a comparative and concentration-resolved analysis of hydrogen-bond environments across different sugars, providing molecular-level insights relevant to aqueous solution structure, biophysical hydration, and liquid-phase carbohydrate chemistry.
Eukaryotic mRNAs typically encode a single functional polypeptide, a principle challenged by the discovery of widespread non-canonical peptide-coding ORFs within 5'UTRs. However, their functional significance at the protein level remains underexplored. Using a four-layered pipeline, we identify 14 human transcripts predominantly transcribed in polycistronic forms, each encoding two conserved proteins. Focusing on the SLC35A4 transcript, we show that its 5'UTR encodes a mitochondrial inner membrane-localized microprotein that we name STREMI (SLC35A4 stress response regulating MICOS interactor). Sharing topology and motifs with the MICOS core subunit MIC10, STREMI regulates mitochondrial cristae morphogenesis in mice and human cells. Additionally, the STREMI-encoding uORF mediates stress-responsive translation of SLC35A4-a Golgi nucleotide sugar transporter-upregulating its translation during the integrated stress response. Evolutionary analyses indicate that these bicistronic transcripts likely arose through transcriptional readthrough following retroposition. We propose a mechanism of "gene symbiosis" that enables functional partitioning and coordinated translation of protein pairs from bicistronic transcripts.
Macromolecular crowding plays a pivotal role in shaping protein stability and bridges insights from in vitro studies to the cellular environment. We investigated the stability of CRABP I through urea melt studies in the presence of PEG 2000 and PEG 4000, monitoring fluorescence wavelength shifts as sensitive indicators of structural transitions. In the absence of crowding agents, CRABP I unfolded with Cm at 4.39 M urea, whereas both PEG variants shifted the unfolding transition to higher concentrations, indicating an enhanced stability. This stabilization reflects crowding-induced reshaping of the free energy landscape, where excluded-volume effects entropically favor the native compact state. PEG 4000, with its larger size, imposed stronger steric constraints and augmented preferential hydration, thereby reinforcing intramolecular interactions and restricting the access of urea to the hydrophobic core. Complementary molecular dynamics simulations corroborated these mechanisms, highlighting how macromolecular crowding governs protein folding pathways and stability under physiologically relevant conditions.