Biochemistry [JOURNAL]
Sun
200 papers
Sphingosine-1-Phosphate Is a Key Signaling Molecule in Normal Conditions and in Multiple Sclerosis.
Ganin DA, Zakharova MN
Biochemistry (Mosc) · 2026 May · PMID 42219383 · Publisher ↗
Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive signaling molecules of sphingolipid metabolism, which plays a pivotal role in regulating numerous processes in the central nervous system and...
Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive signaling molecules of sphingolipid metabolism, which plays a pivotal role in regulating numerous processes in the central nervous system and immune system. Acting as an extracellular ligand for five subtypes of G-protein-coupled receptors (S1PR-S1PR) as well as an intracellular metabolic mediator, S1P controls lymphocyte migration, blood-brain barrier permeability, survival and differentiation of oligodendrocytes, reactivity of astrocytes and microglia, and balance between inflammation, neurodegeneration, and neuroprotection. In pathogenesis of the demyelinative diseases, particularly multiple sclerosis, disruption of the "sphingolipid rheostat" is observed - a shift toward predominance of pro-apoptotic ceramides and relative decrease in the S1P levels, which promotes prevalence of the neuroinflammatory and neurodegenerative processes over remyelination. This review summarizes current data on the structure, metabolism, and intra- and extracellular signaling pathways of S1P, its dual role under physiological conditions and in multiple sclerosis, and analyzes approaches to pharmacological modulation of S1P signaling pathways, highlighting the prospects of selective targeted therapy aimed at immunomodulation, neuroprotection, and stimulation of remyelination.
Integrating Dopamine and BDNF Hypotheses: The Role of Dopamine-BDNF Crosstalk in Neuropathologies.
Alsalloum M, Tsybko A, Naumenko V
Biochemistry (Mosc) · 2026 May · PMID 42219382 · Publisher ↗
The dopamine system plays an important role in numerous physiological processes, such as locomotion, emotions, reward behavior, memory, and learning. Differentiation and proper functioning of dopaminergic neurons are lar...
The dopamine system plays an important role in numerous physiological processes, such as locomotion, emotions, reward behavior, memory, and learning. Differentiation and proper functioning of dopaminergic neurons are largely promoted by the brain-derived neurotrophic factor (BDNF). BDNF is one of the most abundant neurotrophins in the mammalian brain and is implicated in neuronal development, differentiation, and plasticity. Dysfunction of dopaminergic system and BDNF signaling are linked to the pathogenesis of many neurological disorders, including Parkinson's disease, schizophrenia, autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and others. Accumulating evidence indicates a significant crosstalk between BDNF and dopamine system, the disruption of which might underlie the development of various neuro- and psychopathologies. In this review, we summarized recent data on the interplay between BDNF and brain dopamine system under physiological conditions, as well as in neurodevelopmental and neurodegenerative disorders. The available data suggest that the crosstalk between the brain dopamine system and BDNF is essential for normal neurophysiology; it is also involved in neuropathology and could serve as a target for therapeutic strategies aimed at correcting pathological behaviors.
Metabolic Neurophilosophy: Linking Brain Function with Body Metabolism.
Gulyaeva NV
Biochemistry (Mosc) · 2026 May · PMID 42219381 · Publisher ↗
This special issue of Biochemistry (Moscow) "Interaction between Neural Signals and Metabolic Pathways: Role in the Functioning of a Healthy and Diseased Brain", includes studies on the mechanisms of close functional con...
This special issue of Biochemistry (Moscow) "Interaction between Neural Signals and Metabolic Pathways: Role in the Functioning of a Healthy and Diseased Brain", includes studies on the mechanisms of close functional connections between the brain and other organs and tissues of the body. These mechanisms link brain metabolism with its signaling function under normal and pathological conditions. The metabolic signals that enable these connections are the focus of research in this field, which is crucial for an integrated understanding of how the body functions. An impairment in metabolic signaling leads to the development of various pathologies. Metabolites such as glucose, fatty acids, and amino acids act as primary signals that influence neural networks and brain chemistry. This connection between the body's metabolism and brain signaling is not merely a matter of fuel supply, but rather a complex information exchange process. The interaction between the brain and the body occurs within the framework of coordinated work of two main axes: the brain-to-body axis ("from top to bottom" or from center to periphery), and the body-to-brain axis (from periphery to center). This relationship between brain function and body metabolism forms a mechanical and logical connection between metabolic somatic diseases and brain disorders that may underlie their comorbidities. The close connection between brain function, metabolism, and the metabolism of peripheral organs and tissues forms the basis for treating "body-brain metabolic" disorders. Identifying the molecular and cellular mechanisms underlying this relationship allows identifying targets for treating and preventing comorbid somatic and brain conditions. The recent achievements, which prove the close relationship between metabolism and brain activity, have led to the emergence of a rapidly growing interdisciplinary field at the interface of neuroscience, philosophy of consciousness, and functional biochemistry of metabolism. This new synthetic field can be called "metabolic neurophilosophy". Its subject is to explore the integrity and inseparability of the body's metabolism (including both in the brain and peripheral organs and tissues) and the signaling and informational function of the brain. It also studies the dependence of all brain activity, including cognition, and mental states on energy processes and metabolic signaling throughout the body.
The Thiol-Redox Biochemistry of Trypanosomatids: A New Perspective Based on Known and New Actors.
Comini MA, Graña M, Manta B
Biochemistry · 2026 Jun · PMID 42207921 · Publisher ↗
Maintaining thiol redox homeostasis is key for cell viability and development. Therefore, all organisms are equipped with redox systems conformed by redox-active proteins and low-molecular-weight thiols that facilitate t...
Maintaining thiol redox homeostasis is key for cell viability and development. Therefore, all organisms are equipped with redox systems conformed by redox-active proteins and low-molecular-weight thiols that facilitate target-specific delivery of electrons from NADPH to different (macro)molecules. Trypanosomatids are early branching single-cell eukaryotes harboring a unique thiol-redox system centered on the use of trypanothione (-glutathionylspermidine) as a low-molecular-weight thiol-redox cofactor. The irruption of trypanothione in these organisms acted as a major and positive selective pressure that shaped their redox biochemistry. The most radical changes involved the loss of otherwise highly conserved and indispensable glutathione reductase and thioredoxin reductase genes and the concomitant assignment of secondary roles to the related redoxins (glutaredoxins and thioredoxins). Here, we revise the state-of-the-art on the field and provide new research perspectives based on the identification of novel members of the thioredoxin-fold family.
Structural and Biochemical Insights into the Arabinose and Xylose Derivatives of Cyclic-GAMP-Mediated Inhibition of Cyclic-di-AMP Phosphodiesterase.
Hanuman DS, Neeharika S, Nitin K … +3 more , Abhishek S, Sinha KM, Rajakumara E
Biochemistry · 2026 May · PMID 42206963 · Publisher ↗
Bacterial infection, including that of, leads to the accumulation of the bacterial cyclic dinucleotides (CDN), c-di-AMP and c-di-GMP, and the host cGAMP synthase-catalyzed CDN, 2'3'-cGAMP in the cytosol, which activates...
Bacterial infection, including that of, leads to the accumulation of the bacterial cyclic dinucleotides (CDN), c-di-AMP and c-di-GMP, and the host cGAMP synthase-catalyzed CDN, 2'3'-cGAMP in the cytosol, which activates STING-dependent type I interferon (IFN) and NF-κB immune responses. The mycobacterial cyclic dinucleotide phosphodiesterase (CdnP) secreted into the host macrophages blunts host immunity by directly cleaving bacterial- and host-derived CDNs. The arabinose- and xylose-modified 2'3'-cGAMP (2'3'-(A/X)cGAMP) analogues act as potent STING agonists and resist hydrolysis by the host-PDE ENPP1. Here, we report that 2'3'-(A/X)-cGAMP analogues bind to CdnP and compete with its substrate binding. Further studies revealed that these analogues inhibit the catalytic activity of CdnP. The cocrystal structure demonstrates that the arabinose-derived 2'3'-cGAMP (AR-cGAMP) analogue is trapped in an unusual U-shaped conformation in the substrate-binding pocket, away from the catalytic residue and Mn, which suggests that CdnP is incompetent to hydrolyze the analogue and cannot accept the other CDN substrate for hydrolysis. Given that several bacterial and viral pathogens deploy CDN phosphodiesterase enzymes to hydrolyze both host and pathogen-derived STING agonists, sugar-modified CDNs can be used to weaken bacterial and viral defenses and stimulate the STING-mediated host immunity against these pathogens.
Deacetylation of β-Mannans by Two Complementary Carbohydrate Esterases from the Human Gut Microbe .
Lindstad LJ, Mrozek PM, Boehlich GJ … +4 more , Leivers S, Pope PB, La Rosa SL, Westereng B
Biochemistry · 2026 Jun · PMID 42187162 · Full text
β-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several β-mannans are decorated with acetylations, which are key to their physicochemical properties and protection a...
β-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several β-mannans are decorated with acetylations, which are key to their physicochemical properties and protection against enzymatic degradation. Depolymerization of acetylated β-mannans has been described in depth for members of the phylum Bacillota, while there is limited mechanistic knowledge on how Bacteroidota utilizes these glycans. Here, we combined proteomics and biochemical analyses to functionally characterize a pair of carbohydrate esterases (CEs) from that, together, deacetylate complex β-mannans. We demonstrate that the newly identified CE25 enzyme, representing a new carbohydrate esterase (CE) family, exhibits high specificity toward axially oriented 2--acetyl groups on mannose residues. In contrast, CE7 functions as a broad-spectrum esterase, capable of deacetylating oligosaccharides from structurally diverse substrates. Overall, our findings provide new insight into the strategies that beneficial have evolved to deacetylate complex β-mannans in the human gut.
Structural Basis for Potassium Inhibition of WNK Kinases.
Goldsmith EJ, Pleinis JM, Wagner A … +7 more , Mykhaylyk V, Akella R, Humphreys JM, He H, Norrell L, Morrison DE, Rodan AR
Biochemistry · 2026 Jun · PMID 42186973 · Publisher ↗
WNK kinases are chloride- and osmotic-stress-regulated protein kinases recently shown to be controlled by potassium. Prior studies demonstrated the direct binding of chloride and osmotic stress-related water in WNK kinas...
WNK kinases are chloride- and osmotic-stress-regulated protein kinases recently shown to be controlled by potassium. Prior studies demonstrated the direct binding of chloride and osmotic stress-related water in WNK kinase regulation. Here, we probe potassium binding and regulation of WNK kinases via crystallography coupled with mutagenic analysis of WNK kinase autophosphorylation and activity. Crystals of unphosphorylated WNK1 grown in cesium formate, a surrogate for potassium, yielded nonsulfur scattering peaks at 5.75 keV. Mutations were introduced into amino acids flanking the anomalous diffraction peaks. Mutations in WNK1/E388 and the corresponding WNK3/E314, probing a peak close to WNK1/I384, led to reduced inhibition by potassium while maintaining kinase autophosphorylation and substrate phosphorylation activity. Other peaks probed by mutagenesis either did not bear out as potassium regulatory sites or were not validated due to the inactivity of the mutants synthesized. Previously synthesized chloride- and water-binding mutants demonstrate correlated sensitivity to chloride and potassium. Potassium, chloride, and water are all WNK inhibitors that share a common mechanism binding the same low-activity asymmetric dimer of WNK1 kinase domains.
Peptide Mimetic Platform for Activity Profiling of Mycobacterial l,d-Transpeptidases.
Ocius KL, Sanborn RE, Naick A … +2 more , Brammer Basta LA, Pires MM
Biochemistry · 2026 Jun · PMID 42170912 · Full text
Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by () and nontuberculous mycobacteria (NTM). l,d-Transpeptidases (Ldts) are attractive dr...
Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by () and nontuberculous mycobacteria (NTM). l,d-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall cross-linking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor cross-linking. After optimizing assay parameters, we profiled six Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of β-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams, and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.
Mechanisms of Red-Shifted Absorption in Biliverdin-Binding Proteins.
Noji T, Saito K, Ishikita H
Biochemistry · 2026 Jun · PMID 42157484 · Full text
Biliverdin (BV) is a linear tetrapyrrole chromophore that enables far-red and near-infrared photoreception in phytochromes and fluorescence in engineered probes. Here, we systematically investigate a broad set of BV-bind...
Biliverdin (BV) is a linear tetrapyrrole chromophore that enables far-red and near-infrared photoreception in phytochromes and fluorescence in engineered probes. Here, we systematically investigate a broad set of BV-binding proteins using a consistent quantum mechanical/molecular mechanical framework and demonstrate that their absorption wavelengths can be quantitatively reproduced across both BV- and phycocyanobilin (PCB)-binding proteins. In contrast to PCB-binding proteins, BV-binding proteins show a substantially weaker dependence of absorption wavelength on chromophore coplanarity. Instead, the BV color tuning arises from the combined effects of chromophore conformation, electrostatic interactions, desolvation, and π-stacking. Inverted D-ring geometries in Agp2-PCM and Agp2-PAiRFP2 are associated with pronounced red shifts; tryptophan-mediated π-stacking contributes to additional red shifts in JSC1_58120g3-type proteins, and, in sandercyanin, high solvent exposure leads to substantially smaller desolvation-induced blue shifts. Collectively, in contrast to PCB-binding proteins, where absorption trends are largely governed by chromophore shape, BV-binding proteins show relatively stronger contributions from additional factors such as electrostatic interactions and desolvation.
Additional Binding Domain of Marine Chitinase Increases Binding Specificity and Prolongs the Lifetime of Productive Binding State.
Tanimoto H, Tsudome M, Tachioka M … +6 more , Baba A, Miyazaki M, Uchihashi T, Iino R, Deguchi S, Nakamura A
Biochemistry · 2026 Jun · PMID 42150185 · Full text
In aquatic environments, insoluble polymers such as chitin are spatially heterogeneous and rapidly diluted by diffusion, posing a challenge for efficient microbial degradation. A key unresolved question is how extracellu...
In aquatic environments, insoluble polymers such as chitin are spatially heterogeneous and rapidly diluted by diffusion, posing a challenge for efficient microbial degradation. A key unresolved question is how extracellular enzymes remain associated with such substrates long enough to sustain activity. Here, we show that dual binding domains in a marine Chitinase from prolong enzyme residence time on chitin surfaces. Compared with a binding-domain-truncated variant, the full-length enzyme exhibits a significantly lower apparent Michaelis constant at low substrate concentrations, indicating enhanced efficiency, while displaying substrate inhibition at higher concentrations. Single-molecule fluorescence imaging reveals that an additional C-terminal carbohydrate-binding module selectively stabilizes binding to specific crystalline chitin surfaces and increases binding duration by over an order of magnitude. Kinetic simulations based on measured dissociation rates indicate that this prolonged residence time arises from frequent rebinding of the catalytic domain, facilitated by the additional binding module. High-speed atomic force microscopy further shows that this extra domain does not affect the enzyme's movement velocity or processive run length along chitin fibers. Together, these findings demonstrate that selective surface binding and extended binding time, rather than catalytic turnover or motility, are critical determinants of enzymatic efficiency under dilute conditions. Notably, this additional binding domain is common in GH18 chitinases from marine bacteria but absent in those from terrestrial organisms and insects, which operate in high-substrate or low-moisture environments. These results suggest that Chitinase domain architecture has evolved to match environmental conditions, offering new insights into solid polymer degradation.
COSMIC-Linked Ras Mutations at the Interface Between H-Ras and PI3Kγ Frequently Generate Affinity Increases.
Mead EH, Batz KC, Shih KH … +9 more , Fleming IR, Tesdahl CD, Lizardos L, Armendariz JR, Hannan JP, Hickey AM, Leyk A, Erbse AH, Falke JJ
Biochemistry · 2026 Jun · PMID 42148525 · Full text
The three conventional isoforms of the Ras G-protein (H-, K-, N-Ras) function as molecular on-off switches that regulate a wide array of signaling pathways, including the Ras-PI3K-PIP-PDK-AKT pathway that is central to i...
The three conventional isoforms of the Ras G-protein (H-, K-, N-Ras) function as molecular on-off switches that regulate a wide array of signaling pathways, including the Ras-PI3K-PIP-PDK-AKT pathway that is central to innate immunity and normal cell growth and is dysregulated in many disease states. Activation of the pathway by Ras requires adequate Ras-PI3K binding affinity. Here we focus on the interface of known structure in the H-Ras:PI3Kγ co-complex essential to multiple pathways including directed pseudopod growth in leukocyte chemotaxis. At this interface 10 H-Ras residues, all 100% conserved between the H-, K-, and N-Ras isomers, contact the Ras binding domain of PI3Kγ (PI3Kγ). To investigate the degree to which the native H-Ras:PI3Kγ interface is optimized by evolution for maximal binding affinity, 8 interfacial Ras mutations selected from the COSMIC database and the literature were introduced at the contact positions. All 8 Ras mutations were observed to alter the H-Ras:PI3Kγ binding affinity, with 4 mutations yielding significant affinity increases and 4 yielding significant affinity decreases. These findings indicate that the native H-Ras:PI3Kγ interface provides intermediate, rather than maximal, binding affinity. Such intermediate affinity is consistent with the substantial binding plasticity of the conserved H-, N-, K-Ras effector docking surface, which has evolved to bind a diverse array of effectors. Furthermore, the findings provide evidence that COSMIC-linked mutations at the H-Ras:PI3Kγ interface frequently generate affinity increases (not just the affinity decreases typical of random interfacial mutations) with potential implications for molecular mechanisms of disease and for tool development in cell biology.
Dynamic Pathway Selectivity of TAS2R5 toward or Away from β-Arrestin or G Protein from Biased Agonists.
Kim D, Strzelinski HR, Longabardi CM … +1 more , Liggett SB
Biochemistry · 2026 Jun · PMID 42142023 · Full text
Bitter taste receptors (TAS2Rs) are GPCRs functionally expressed in extraoral organs including the lung, heart, brain, and gastrointestinal system and are candidate targets for novel drugs. It is unclear whether TAS2R5 c...
Bitter taste receptors (TAS2Rs) are GPCRs functionally expressed in extraoral organs including the lung, heart, brain, and gastrointestinal system and are candidate targets for novel drugs. It is unclear whether TAS2R5 can be stabilized by structurally distinct agonists resulting in pathway selectivity (biasing) to achieve optimal outcomes. We screened TAS2R5 agonists for signaling toward or away from β-arrestin or G protein ([Ca]). Agonists T5-1 (1,10-phenanthroline) and T5-6 (4,7-dimethyl-1,10-phenanthroline) coupled equally to G protein but showed marked differences in homologous desensitization (∼21% and ∼91%, respectively). This desensitization was due to dissimilar degrees of β-arrestin engagement compared to the reference agonist T5-7: T5-6 evoked up to ∼86% enhancement of β-arrestin1 or β-arrestin2 recruitment, while T5-1 was biased in the opposite direction, up to ∼78% less, compared to T5-7. T5-1 elicited little receptor internalization compared to T5-6 consistent with the β-arrestin findings. For the initial response, T5-1 and T5-6 represent one-pathway differences (decreased or increased β-arrestin) leading to bias in different directions, while G protein signaling was equivalent. However, under desensitizing conditions T5-6 was decoupled from G protein representing two-pathway biasing in opposing directions for T5-6 (increased β-arrestin and decreased G protein), imposing extreme biasing of β-arrestin over G protein. Our finding that agonists can affect β-arrestin biasing in either direction suggests that TAS2R5 is sufficiently pliable for specific signals to be engineered depending on the desired therapeutic outcome. The dynamic nature of the directionally contrasting changes of the two pathways over time accentuated bias and is due to the interaction of the two signals.
Dimerization of MilM Is Essential for Catalyzing the Pyridoxal-5'-phosphate (PLP)-Dependent Cγ-Hydroxylation of l-Arginine during Mildiomycin Biosynthesis.
Das S, Naik Y, Mishra U … +4 more , Ganguly M, Dubey BN, Jana S, Mahanta N
Biochemistry · 2026 Jun · PMID 42136426 · Publisher ↗
MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as l-arginine oxidase cum -hydroxylase. Here, we report detailed b...
MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as l-arginine oxidase cum -hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics (MD) simulation-based investigations of MilM from B-98891 to elucidate the mechanisms of substrate binding and catalysis and the roles of the active-site residues involved in these processes. Our experimental results confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the l-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid via a possible superoxide radical anion intermediate, while generating HO and NH as reaction byproducts. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. Structural modeling, MD simulation, and site-directed mutagenesis reveal that residues from both protomers contribute to cofactor and substrate stabilization, including Lys232 and His31 as key catalytic residues as well as additional residues forming an extended active-site interaction network. Moreover, MD simulations uncovered a dimer-mediated alternating-lid mechanism in which coordinated motions at the dimer interface regulate substrate access and product release through transient tunnels while maintaining a protected catalytic environment. This seesaw-like dynamics provides a mechanistic framework for understanding how dimerization controls enzyme function, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into substrate/cofactor stabilization and the catalytic mechanism of MilM and expand our understanding of the remarkable PLP-dependent oxidases and hydroxylases in natural product biosynthesis.
Integrative Modeling Identifies a Dual-Function Helix in Full-Length MDM2, Providing Novel Mechanistic Insights.
Chaudhary H, Pandey A, Chowhan RK
Biochemistry · 2026 Jun · PMID 42130315 · Publisher ↗
The E3 ubiquitin ligase MDM2, an important oncogene, is a central negative regulator of p53, yet its extensive intrinsically disordered regions have hindered structural characterization of the full-length protein. Here,...
The E3 ubiquitin ligase MDM2, an important oncogene, is a central negative regulator of p53, yet its extensive intrinsically disordered regions have hindered structural characterization of the full-length protein. Here, we integrate AlphaFold ensemble modeling to sample the conformational landscape with all-atom molecular dynamics simulations to characterize structural stability and construct a comprehensive, dynamic model of full-length MDM2. Our analysis identifies a previously uncharacterized autoinhibitory helix (upstream of the central acidic domain) in MDM2 that dynamically masks the N-terminal p53-binding pocket. The dynamic ensembles reveal a multistep unmasking pathway in which the helix pivots away to expose the buried p53-binding pocket. This is followed by a stabilization mechanism whereby, in the bound complex, the same helix repositions to act as an adaptive clamp, anchoring the docked p53. Our study indicates this dynamic gating apparatus to be evolutionarily conserved among MDM2 homologues. These findings provide an atomic-level framework for understanding how MDM2's intrinsic dynamics regulate p53 recognition and suggest new avenues for therapeutic targeting of the p53-MDM2 axis.
A Miniaturized, High-Throughput Aqueous Solvent-Centric Method for Protein Solubility Screening.
Svoboda A, Molineris M, Tureckiova T … +3 more , Hlouchová K, Pluskal T, Hebra T
Biochemistry · 2026 Jun · PMID 42118572 · Full text
Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe an aqueous, solvent-centric, fully miniaturized 96-well workflow to screen extraction conditi...
Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe an aqueous, solvent-centric, fully miniaturized 96-well workflow to screen extraction conditions that preserve soluble recombinant protein during lysis and clarification in a single working day. Liquid-nitrogen-frozen pellets are cryogenically bead-milled with stainless-steel beads, retaining the native intracellular milieu while ensuring uniform disruption. The resulting wet, frozen cell powder can therefore be extracted with user-defined solvent, enabling systematic exploration of pH, ionic strength, detergents, and chaotropes. Protein solubility is assessed by a 1 μL chromogenic anti-His dot-blot. We demonstrate the use of the protocol by solubilizing a set of highly challenging -generated proteins and show that dot-blot intensity provides a practical semiquantitative proxy for successful extraction of soluble proteins. We also provide experimentally supported guidelines on the influence of solvent reagents on subsequent steps of protein production, SDS-PAGE, and Ni-NTA purification. This workflow is compatible with upstream genetic solubility-enhancement, chassis- and cultivation-based strategies and enables direct transition from screening hits to scale-up. Because the workflow uses standard molecular biology equipment and inexpensive consumables, it can be readily adopted or automated in most laboratories.
Structure-Based Design of UVRAG-BAX Binding Interface-Targeting Compound Induces Apoptosis and ROS in Breast Cancer Cells.
Kumar S, Singh RB, Dubey VK
Biochemistry · 2026 May · PMID 42117566 · Publisher ↗
Targeting protein-protein interaction interfaces has emerged as an effective strategy in modern anticancer drug discovery, as these interfaces regulate critical signaling pathways involved in tumor progression and surviv...
Targeting protein-protein interaction interfaces has emerged as an effective strategy in modern anticancer drug discovery, as these interfaces regulate critical signaling pathways involved in tumor progression and survival. In this study, a structure-based strategy was used to identify potential small-molecule inhibitors targeting the interaction interface of the UVRAG-BAX protein complex. Initially, the interaction interface between UVRAG and BAX was characterized using protein-protein docking. Subsequently, virtual screening of compounds from the ZINC database was carried out, followed by molecular docking to identify molecules capable of binding to the targeted interface region. Cytotoxicity analysis using the MTT assay showed a potent inhibitory effect with an IC value of 5 ± 0.283 μM. Furthermore, mechanistic studies suggested that treatment with the compound significantly increased the intracellular level of reactive oxygen species causing apoptotic cell death, arrested the cell cycle in S phase and elevated the Sub G population in MCF-7 cells. Confocal microscopy using AO/DAPI staining further confirmed cell death in treated cells. These findings suggest that 1-[2-(4,11-dimethyl-2-oxo-6,7,8,9-tetrahydro-[1]benzofuro[3,2-]chromen-3-yl)acetyl]-4-phenylpiperidine-4-carboxylic acid (ZINC000002107582) is a promising lead compound that targets the UVRAG interaction interface and exerts potent anticancer effects in breast cancer cells.
Keeping Wavering Bonds: Deactivation-Induced Signaling by Reactive Electrophiles.
Long MJC, Karakoç Y, Aye Y
Biochemistry · 2026 Jun · PMID 42117458 · Full text
Discovered ∼60years ago, the lipid metabolite 4-hydroxynonenal (HNE) is linked to a plethora of macromolecular targets and biological functions. For a molecule that weighs 156 Da and possesses a single H-bond donor, this...
Discovered ∼60years ago, the lipid metabolite 4-hydroxynonenal (HNE) is linked to a plethora of macromolecular targets and biological functions. For a molecule that weighs 156 Da and possesses a single H-bond donor, this is quite a feat. Despite its chemical simplicity, HNE contains an α,β-unsaturated aldehyde system, endowing it with the capability to react covalently with numerous biological functional groups and bestowing on it pleiotropic properties. Regardless of the specific entity engaging with HNE, it is covalent bond formation that has dominated thought on HNE behavior. Indeed, cells possess a flurry of detoxifying enzymes that convert HNE to less reactive chemicals lacking the α,β-unsaturated aldehyde. For instance, the cell can either reduce or oxidize the aldehyde within HNE, deactivating HNE's chemical reactivity. Here, we discuss one of our recent papers that discovered that HNE can modify the detoxification enzyme, Cyp-33e1, in , using a customized tissue-specific screen for HNE-sensor proteins. Consistent with the concepts of active site partitioning, HNE also emerged as a substrate of Cyp-33e1. We next discovered that HNE changed lipid storage in worms in a Cyp-33e1-dependent manner. We proposed that the product of Cyp-33e1 detoxifying HNE was responsible for this change in lipid storage and were able to show that 4-hydroxynonenoic acid (HNA), the product of Cyp-33e1 oxidation of HNE, causes this phenotype. We have dubbed this new signaling mode, "deactivation signaling". It sets an important precedent for how the bioactivity of HNE is considered, and we discuss the ramifications of this result in this perspective.
Disordered N-Terminal Tail "Wags the Dog" in Human Thymidylate Synthase.
Ricciardi MN, Sapienza PJ, Lee AL
Biochemistry · 2026 May · PMID 42114025 · Full text
The utility of intrinsically disordered regions (IDRs) in protein function has become an area of broad interest in recent years given their known roles in signaling, biomolecular condensates, and protein activity regulat...
The utility of intrinsically disordered regions (IDRs) in protein function has become an area of broad interest in recent years given their known roles in signaling, biomolecular condensates, and protein activity regulation. The central challenge, however, is that disorder is difficult to characterize, and thus, there is a need to develop approaches to monitor how IDRs influence proteins. Here, we employ a strategy to restrict the freedom of the intrinsically disordered N-terminal tail (ID-tail) of human thymidylate synthase (hTS), which, together with tail truncation, enables testing aspects of how the ID-tail modulates enzyme function. The highly flexible hTS tail is known to impact allosteric substrate cooperativity and conformational switching dynamics, although how the tail brings about these effects has not been determined. We attempted to restrict native tail dynamics by chemically linking the dimer tails and tested linked forms with activity, binding, and nuclear magnetic resonance heteronuclear single quantum coherence (NMR HSQC) spectra. Truncating four N-terminal residues eliminated cooperativity in dUMP binding to apo-hTS, but because a similar result was obtained from the linked full-length protein, complete tail freedom appeared to be important for intersubunit communication. NMR analysis was aided by referencing the inactive M190K mutant, which roughly defined regions of active-inactive conformational switching that were disrupted by tail linkage. Collectively, these results point toward a role of the ID-tail to influence the ordered core of the protein. Moreover, while the natural ID-tail is extremely flexible, it likely possesses specific physiochemical movement dynamics and positioning attributes that facilitate its impact on allosteric cooperativity and conformational dynamics.
Correction to "Model Peptides Enable Comparisons of Substrate Binding Modes of Hsp70 Molecular Chaperones".
Bhasne K, Dörries L, Maqtedar A … +3 more , Mager ST, Clerico EM, Gierasch LM
Biochemistry · 2026 May · PMID 42101097 · Full text
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Cyclophilin A Induces Mechanisms of Cell Rearrangement and Fusion During Differentiation and Maturation of Early Macrophage Precursors.
Kalinina AA, Alexandrova AY, Kazansky DB … +1 more , Khromykh LM
Biochemistry (Mosc) · 2026 Apr · PMID 42070983 · Publisher ↗
Macrophages are a heterogeneous cell population whose functional diversity is formed during their maturation and depends on factors of the microenvironment after their migration into the bloodstream or tissues. One such...
Macrophages are a heterogeneous cell population whose functional diversity is formed during their maturation and depends on factors of the microenvironment after their migration into the bloodstream or tissues. One such factor is the pro-inflammatory protein cyclophilin A (CypA, 18 kDa). Using a model of early human monocytic THP-1 cells, it was shown that recombinant human CypA (rhCypA) exerts a differentiating effect on these cells, inducing their maturation, adhesion, and spreading. Under the effect of rhCypA, the THP-1 cells developed an actin cytoskeleton characteristic of motile cells with numerous pseudopodia and podosomes, which ensure tight adhesion of the cells to the substrate and determine their migratory capabilities. Combination of low concentrations of rhCypA and other activators (phorbol myristate acetate) showed an additive effect and ensured effective monocyte differentiation. It was shown that rhCypA, along with other pro-inflammatory factors (IFNγ, TNFα), promotes cell fusion and induces formation of multinucleated macrophages, which are formed during osteoclast maturation under normal conditions as well as during granuloma formation in chronic inflammation (tuberculosis, Crohn's disease). Multinucleated giant cells have significantly higher functional activity (phagocytosis, bactericidal, and pro-inflammatory activity) compared to the mononuclear forms. The study showed that rhCypA enhances expression of the CD147 molecule, an integral functional regulator of CD29 and CD98 molecules involved in the processes of cell adhesion and fusion. Elevated doses of CypA cause deterioration in macrophages, inducing their apoptosis, which may play a role in regulation of the immune response. The findings of this study determined the mechanisms by which secreted CypA mediates monocyte differentiation and maturation, as well as it showed functional role of macrophages in the development of the immune response, which could facilitate further development of therapeutic approaches for the treatment of infectious, autoimmune, and other diseases.
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