Parkinson's disease (PD) is characterized by the pathological aggregation of α-synuclein (α-syn) into β-sheet-rich fibrils, contributing to neuronal toxicity and oxidative stress. In this study, we investigated the inhibitory and disaggregating effects of Triprolidine (TC) on α-syn fibrillation through a combined experimental and computational approach. Biophysical assays, including ThT assay, DLS, and ANS assays, demonstrated that TC inhibits α-syn fibrillation in a concentration-dependent manner (IC50 ≈ 255 μM), disrupts preformed fibrils, and maintains the protein's native form. CD data further revealed that TC prevents the transition of α-syn to its toxic β-sheet-rich form and facilitates partial structural reversal during disaggregation. To elucidate the molecular mechanism of inhibition, we performed all-atom molecular dynamics (MD) simulations followed by Markov State Model (MSM) construction. The simulations revealed that TC binding remodels the conformational landscape of α-syn by stabilizing compact, disordered states and reducing the population of β-sheet-prone intermediates, particularly in the aggregation-prone NAC region. MSM analysis identified metastable states with diminished aggregation potential and reduced inter-residue contact probability, offering mechanistic insights into how TC interferes with early nucleation events. TC attenuates seeded fibrillation in a concentration-dependent manner too. Complementary cellular assays, including MTT and hemolytic assays, confirmed a significant reduction in α-syn-induced cytotoxicity upon TC treatment, with a decrease in ROS levels as confirmed by the DCFH-DA assay. Together, these findings demonstrate that TC modulates both the structural dynamics and functional toxicity of α-synuclein, and highlight its potential as a promising chemical modulator for further investigation in PD-related protein aggregation.

Cryptochromes act as photoreceptors in diverse organisms and bind flavin adenine dinucleotide (FAD) as a chromophore. The plant-like cryptochrome CryP from the diatom Phaeodactylum tricornutum regulates in vivo the expression of light-harvesting proteins in response to blue light. CryP carries, in addition, 5,10-methenyltetrahydrofolate (MTHF) for light capturing. In contrast to most other cryptochromes, FAD in CryP is present as a stable flavin neutral radical in the dark, which undergoes a photoreduction to form the fully reduced state in the light. Here, we demonstrate by applying nanosecond-time-resolved UV-vis spectroscopy that the flavin neutral radical is photoreduced within 100 ns to the fully reduced state and subsequently recovers very fast with a time constant of 1.4 ms in the absence of reducing agents or in the presence of 1 mM dithiothreitol. Despite its short lifetime, the transient light state is sufficient to induce homo-oligomerization of CryP as shown by light-dependent size exclusion chromatography. A long-lived fully reduced flavin is only formed in the presence of external reducing agents by a second pathway that is lost after a single full conversion, likely by degradation. Then, the recovery to the neutral radical state takes hours with a time constant of 40 min. Moreover, we validate the existence of energy transfer between the two chromophores depending on the flavin redox state using fluorescence spectroscopy. We propose a detailed mechanism for the photocycle of CryP, highlighting the contribution of two separate reduction pathways.

Adaptor protein AcrA plays a central role in the assembly and function of tripartite multidrug efflux pumps in Gram-negative bacteria, yet how its structural organization responds to coupled chemical perturbations rather than solely to equilibrium conditions remains unclear. Residues near His285 define a hinge microenvironment linking the lipoyl and β-barrel domains, suggesting a site for chemically sensitive structural modulation. Here, site-directed spin labeling combined with continuous-wave electron paramagnetic resonance spectroscopy was used to examine AcrA under an Mg2+-driven perturbation that simultaneously alters proton availability. Mg2+ addition produced spectral broadening at residue 62 that was fully reversed by spin dilution, indicating increased interspin proximity without changes in intrinsic side-chain dynamics. In contrast, direct acidification to a comparable bulk pH in the absence of Mg2+ did not reproduce this behavior. Structural mapping places residue 62 in proximity to the His285-centered hinge region, suggesting that coupled changes in protonation and metal coordination bias local interaction networks and modulate interdomain organization. These findings demonstrate that equivalent bulk conditions can mask distinct molecular states and identify chemical pathways as an important determinant of AcrA structural dynamics.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide-containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the β-sheet region of the B1 domain of protein G (GB1) as well as β-strand interactions in amyloid fibrils of the Parkinson's disease protein α-synuclein. In both contexts, we observe dramatic destabilization of the β-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of α-synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen-bonding networks and prompt future study of both systems.

Sequence-specific recognition of complex, folded RNA structures is a highly desirable yet formidable goal. The present study explored nucleobase-modified peptide nucleic acids (PNAs) as ligands that bind and recognize junctions between single- and double-stranded RNA via Watson-Crick and Hoogsteen hydrogen bonding, respectively. The results showed that these hybrid PNAs exhibited strong affinity for RNA junctions and transitioned from duplex to triplex binding modes without additional modification. However, depending on the sequence context, extending the PNA's backbone at the transition site could yield a slight improvement in binding affinity. The overall binding affinity was modest and comparable to that of triplex-only binding. The duplex-triplex binding mode exhibited relatively low sensitivity to mismatches adjacent to the transition site, suggesting that the transition might be dynamic and not well organized. Overall, the results demonstrated PNA's ability to recognize single-double-strand junctions in RNA; however, the modest stability and specificity might limit this binding mode to specific cases where simpler duplex or triplex binding modes are not feasible.

Molecular glues (MGs) stabilize protein-protein interactions (PPIs) through interactions at composite binding interfaces, thereby promoting cooperative ternary complex formation. For hub proteins that engage in multiple PPIs with widely varying intrinsic affinities, the interplay between binary PPI affinity and MG cooperativity is therefore a key determinant of selective stabilization. Here, we use the multiclient 14-3-3 scaffold protein as a model system to systematically dissect the relationship between binary 14-3-3/client affinity (KDI) and MG-induced cooperativity (α). Client peptide affinity was systematically tuned by modifying residues N-terminal to the phosphorylated 14-3-3 binding motif while preserving the C-terminal composite interface required for MG recognition. Using a combination of biophysical techniques and protein crystallography, we show that changes in KDI alter the thermodynamic and kinetic parameters of both binary and ternary complex formation, but do not affect MG cooperativity. This principle was observed for the noncovalent MG fusicoccin-A as well as covalent MGs targeting 14-3-3σ/client complexes. Competitive binding experiments and thermodynamic modeling further revealed that, although α is independent of KDI, the interplay between KDI, MG affinity (KDII), and cooperativity determines which PPIs are preferentially stabilized in a multiclient environment. Together, these findings establish cooperativity, intrinsic PPI affinity, and MG affinity as key parameters governing MG activity and selectivity, providing a framework for the rational design of MGs targeting hub protein interactomes.

Strategic engineering of natural product biosynthetic pathways through the incorporation of alternative, tunable carbon-based building blocks represents a promising approach for accessing medicinally relevant molecules. However, efforts toward this goal have been hindered by the substrate specificity of component enzymes. In type I and type II fatty acid synthases (FASs) and polyketide synthases (PKSs), the acyltransferase (AT) selects a specific malonyl-based coenzyme A (CoA) building block and transfers it onto the acyl carrier protein (ACP) for subsequent processing. Inspired by the observation that some ACPs can bypass the AT and "self-acylate", we herein explored the tolerance of FAS and PKS ACPs to load both a variety of CoA substrates and ethane thioester (ET) analogs serving as truncated CoA building blocks. We observe that the Escherichia coli (E. coli) AT, FabD, can load and transfer methylmalonyl-CoA (mm-CoA) and malonyl-CoA (m-CoA) onto three ACPs: the type II Streptomyces coelicolor actinorhodin PKS ACP (ActACP), the E. coli type II FAS ACP (AcpP), and the type I Saccharopolyspora erythraea 6-deoxyerythronolide B PKS ACP6 (DEBS ACP6). Synthesized ET analogs of mm-CoA and m-CoA were loaded onto all three ACPs through FabD-assisted acylation. Additionally, both in the presence and absence of FabD, ACPs could be acylated with ET analogs of fluoromalonyl-, succinyl-, and glutaryl- building blocks. Overall, this work pushes the limits of ACP substrate loading, revealing new complexity in carbon-based building block selection and establishing foundations for novel routes toward diverse functional group incorporation in FAS/PKS biosynthetic pathways.

In this work, we report the discovery of a G-quadruplex DNA-selective and near-infrared (NIR-I) emissive G-quadruplex binder derived from a known styryl-quinolinium-based compound. This molecule is unique in its class, with an emission wavelength above 800 nm, and displays selective G-quadruplex binding. Among the synthesized molecules, compound 2 emerged as a selective and the best G-quadruplex binder through comparative studies and exhibited strong thermal stabilization on Pu22 G-quadruplex with a ΔTm of 21.7 °C (at a 1:3 DNA to ligand ratio). G-quadruplex DNA-binding studies showed that thermal stabilization was topology-dependent. CD, UV-vis, and docking studies were performed to confirm the binding of these compounds to the different G-quadruplexes. Solution NMR studies confirmed the binding of compound 2 to the Pu22 G-quadruplex DNA. Cell-based studies showed that compound 2 was significantly cytotoxic to MCF-7 cancer cells and was readily taken up by the cells. However, cell internalization studies showed contrasting changes in the internalization properties, with compound 2 displaying a significant population on the nuclear periphery, which was altogether different from its parent compound 1, which did not show such changes.

The oxidative modification of low-density lipoprotein (LDL) is involved in the generation of lipid peroxidation-derived electrophilic aldehydes, which covalently react with apolipoprotein B-100 to form various adducts. Although the oxidative modification of LDL is critical in the pathogenesis of atherosclerosis, the structural properties of adducts in oxidized LDL (oxLDL) remain unclear. Thus, we aimed to characterize oxLDL using the adductome approach, a comprehensive mass spectrometry-based analysis designed to detect specific product ions from positively ionized oxidized phosphatidylcholine (oxPC) adducts. The amounts of several adducts, including four major oxPC-lysine (oxPC-Lys) adducts, prominently increased when LDL was oxidized with Cu2+. Analysis of the synthetic adduct candidates through mass spectrometry revealed two oxPC-Lys Schiff base adducts and two novel oxPC-Lys amide-type adducts as the major lysine adducts in oxLDL. The oxPC-Lys Schiff base adducts could be transformed into stable amide-type adducts during LDL oxidation. The amounts of oxPC-Lys adducts were significantly higher in the sera of hyperlipidemic mice than in the sera of control mice. The recognition of oxPC-Lys adducts by macrophages suggested that these adducts were involved in the phagocytosis of oxLDL by macrophages. These findings provide insights into the structural properties of lipoproteins modified under oxidative stress and their biological implications. Moreover, this study is the first to identify and quantify oxPC-Lys amide-type adducts in vitro and in vivo.

The cleavage of the C10-C4a bond in anthraquinones is a key step in generating ring-opened quinone derivatives in filamentous fungi. GedF, a short-chain dehydrogenase/reductase (SDR) from Aspergillus terreus, together with the dioxygenase GedK, mediates this transformation, yet the enzymatic mechanism of GedF remains unclear. Here, isotope labeling experiments confirm that reduction of Questin-to-Questin hydroquinone incorporates one proton from NADPH and one from water. Structural modeling, molecular docking, and site-directed mutagenesis reveal that GedF employs a noncanonical catalytic architecture featuring a conserved Ser-Tyr catalytic core instead of the classical Asn-Ser-Tyr-Lys tetrad typical of SDRs. Notably, mutagenesis and comparative analysis indicate that a positively charged residue is required for catalysis but is not strictly position-conserved, consistent with a role in maintaining the catalytic microenvironment and facilitating proton transfer. Phylogenetic and sequence analyses show that GedF belongs to the NAD(P)H-dependent SDR clade, and that variation in the positioning of basic residues occurs among homologues while preserving the conserved Ser-Tyr catalytic core. These findings elucidate the catalytic mechanism of GedF and uncover an alternative SDR catalytic strategy involved in anthraquinone ring-opening biosynthesis in filamentous fungi.

Serine, a nonessential amino acid classically defined as a precursor for protein synthesis and one-carbon metabolism, is increasingly recognized as a signaling metabolite that links the cellular metabolic status to regulatory decision-making. Intracellular serine availability is shaped by nutrient conditions, glycolytic flux, and activity of the serine synthesis pathway, and these fluctuations are sensed to elicit coordinated metabolic and signaling responses. This review discusses mechanisms by which serine modulates cell growth and stress responses, with particular emphasis on its interaction with central nutrient-sensing pathways, including mTORC1 and the integrated stress response. In parallel, serine-driven one-carbon metabolism is examined for its role in supporting nucleotide biosynthesis, methylation reactions, and redox homeostasis through folate-dependent pathways and NADPH generation, thereby coupling anabolic processes to the maintenance of redox balance and genome integrity. In addition to intracellular functions, serine contributes to intercellular signaling. Conversion of l-serine to d-serine mediates neuromodulatory activity via N-methyl-d-aspartate receptors, while serine availability also influences immune cell function, inflammatory signaling, and host-microbe interactions. Dysregulation of serine metabolism and signaling is further considered in the context of disease states, including cancer, neurodegeneration, and metabolic disorders. Together, these observations support a framework in which serine functions as an information-bearing metabolic signal that coordinates the biosynthetic capacity with cellular adaptation and intercellular communication.

Fluorinated succinate analogues were evaluated as mechanistic probes of Mycobacterium tuberculosis isocitrate lyase (MtICL). However, 2,2-difluorosuccinate (1; Ki = 6.1 mM) and 2,2,3-trifluorosuccinate (2; Ki = 23.5 μM) act as reversible noncompetitive inhibitors and meso-2,3-difluorosuccinate (3) displayed slow-onset reversible inhibition (Ki = 30 μM), the 2-fluorosuccinate enantiomers ((R)-4 and (S)-4) produced time-dependent irreversible inactivation. Inactivation by 4 was observable under turnover conditions in the presence of glyoxylate and succinate, consistent with a two-step kinetic mechanism. The S enantiomer inactivated more efficiently than (R)-4, consistent with stereoelectronic alignment required for elimination of HF following abstraction of the pro-S proton. 1H NMR analysis detected maleate formation from (S)-4, and mass spectrometry revealed a +132 Da adduct consistent with covalent modification of Cys191. Notably, kinact/KI values for 4 exceeded that measured for maleate, indicating that covalent capture occurs from an enzyme-bound intermediate prior to product release. These results support a mechanism in which fluorine substitution redirects the enolate-generating half-reaction of MtICL toward elimination and covalent modification. (S)-2-Fluorosuccinate therefore represents a succinate-analogue mechanism-based inactivator that exploits a catalytic step distinct from previously described isocitrate-analogue inhibitors.

The ubiquitous flavoenzymes typically function as oxidoreductases that comprise several distinct main types, including flavoprotein oxidases (FPOs), flavoprotein dehydrogenases (FPDs), and flavoprotein monooxygenases (FPMOs). FPOs and FPDs catalyze two-electron oxidation reactions of organic substrates, typically dehydrogenations, thereby converting oxidized flavin (Flox) into its fully reduced state (Flred). Prior to the next catalytic cycle, molecular oxygen (=dioxygen or O2) or (protein-bound) cofactors facilitate the required Flred reoxidation for FPOs and FPDs. Remarkably, members of these two flavoenzyme types can be homologous with highly similar amino acid compositions and overall structures, as minor protein alterations, particularly in the vicinity of the flavin cofactor, can drastically affect O2 reactivity. Finally, FPMOs incorporate one O2-derived oxygen atom into their substrate. To this end, required electrons for Flred formation and O2 activation either come from NAD(P)H (external FPMOs) or, more rarely, the substrate itself (internal FPMOs). External FPMOs steer O2 reactivity toward the formation of covalent flavin-oxygen adducts primarily at the C4a atom of the flavin's isoalloxazine ring or, in some cases, at the adjacent N5. In contrast, typical internal FPMOs forego the formation of covalent oxygen adducts entirely, although an exception in the form of a flavin-N5-oxide-forming enzyme has been reported. Consequently, natural selection has led to three distinct O2 reactivity patterns in flavoenzymes, which either suppress (FPDs), stimulate (FPOs), or steer (FPMOs) this challenging process. In this review, current knowledge on the relationship between flavoenzymes and O2 is summarized, emphasizing strategies to insert oxygen into organic substrates and counteract uncoupling, while also highlighting open questions and future challenges.

To understand the differences resulting from the presence of an α- or β-amino acid in a peptide, we used two sets of amino acids corresponding to Asp/isoAsp and Ala/isoAla in two types of host peptides: one containing Gly (with no side chains) and the other containing Ala (representing amino acids with side chains), and measured their tendency to form fibrils. The peptides with a β-amino acid formed fibrils, while those with an α-amino acid did not exhibit this proclivity, as inferred from the thioflavin T (ThT) fluorescence intensity measurements. The fibrillation of the hexapeptide with isoAsp (the β-amino acid corresponding to Asp) was inhibited by protein-l-isoaspartyl methyltransferase (PIMT), a repair enzyme that converts the abnormal isoAsp residue to normal Asp. Isothermal Titration Calorimetry revealed the exothermic mode of binding of A6-isoAsp (Ala-based host hexapeptide) with PIMT. Far-UV CD spectroscopy revealed a β-sheet to α-helix transformation of isoAsp-containing peptides in the presence of PIMT. The hydrophobicity of the peptides was measured by noting their distribution in a mixture of water and octanol, where the β-amino acid exhibits a more positive hydrophobicity value than the α-amino acid. This may suggest that the hydrophobic forces bring chains containing β-amino acids together to enable nucleation for fibril formation. Molecular dynamics (MD) simulations indicated that isoAsp-based host peptides, formed into fibrils, have stable structures over the course of simulations, whereas the equivalent peptide with Asp disintegrated. Models of a single β-strand of these peptides bound to the active site of the enzyme were stable.