Biochemistry Biochemistry最新文献

The misfolding of α-synuclein (α-Syn) is a pivotal event in the degeneration of dopaminergic neurons and the progression of Parkinson’s disease. Given its pathological significance, elucidating the self-assembly of α-Syn and developing inhibitors that suppress aberrant misfolding are imperative for effective synucleinopathy therapies. Building upon the remarkable potential of Whitlock’s caffeine-armed molecular tweezer in inhibiting amyloid-β aggregation, this study employs all-atom MD simulations under NPT conditions to explore its impact on α-Syn misfolding. Analyses of the secondary structure and cluster conformations reveal a global transition of the N-terminal and NAC regions into largely unstructured conformations interspersed with multiple β-sheet formations spanning both regions. The simulations further capture the emergence of a β-hairpin structure spanning residues 38–53, a region previously identified as the primary nucleation site for aggregation. Notably, the introduction of the caffeine-tweezers significantly reduces the formation of ordered β-sheet structures. Contact maps, free-energy landscapes, and binding evaluations collectively demonstrate a strong binding preference of the tweezer for the N- and C-terminal regions of the peptide. By engaging in π-stacking interactions with aromatic residues at the termini, the tweezer induces a looped conformation that disrupts non-native contacts between the N-terminus and the NAC segment. This rearrangement restores native long-range interactions between the terminal domains, thereby re-establishing the protein’s intrinsic regulatory mechanism that suppresses NAC-mediated pathological aggregation. These findings elucidate the inhibitory role of the caffeine-tweezer, underscoring its therapeutic potential in targeting α-Syn misfolding. Our findings offer a rational framework for the design of novel therapeutics combating synucleinopathies.

Targeted protein degradation (TPD) technology centered on proteolysis-targeting chimeras (PROTACs) has become an increasingly transformative paradigm in drug discovery. PROTACs, by association with a disease-related target protein of interest and an E3 ligase, form a ternary complex in which the target protein undergoes subsequent ubiquitination and proteasomal degradation. This unique event-driven mechanism of action underscores the importance of kinetic simulation in facilitating the understanding of the kinetic parameters in TPD processes within a kinetic context to guide PROTAC design and optimization. Here, we employ KinTek Explorer to develop kinetic models for simulating PROTAC-induced ternary complex formation and the subsequent mechanistic steps leading to TPD. We illustrate the effects of, and interplay between, PROTAC binding specificity, affinity, cooperativity, and mechanism in complex TPD scenarios. Our findings highlight the effectiveness of KinTek Explorer in TPD kinetic simulation to facilitate PROTAC design.

Zipper-interacting protein kinase (ZIPK) is a ubiquitous serine/threonine protein kinase that plays pivotal roles in regulating cell motility, division, and smooth muscle contractility through phosphorylation of myosin. In this study, we systematically investigated the phosphorylation reactions of smooth muscle myosin (SMM) by ZIPK. We found that ZIPK phosphorylates MRLC sequentially, first at Ser¹⁹ and then at Thr¹⁸, determined by quantitative mass spectrometry analysis on wild-type MRLC. Analysis on phosphomimic and unphosphorylatable MRLC mutants indicates that the phosphorylation rate at Ser¹⁹ on unphosphorylated MRLC is 1.5 times faster than that at Thr¹⁸ on Ser¹⁹-phosphorylated MRLC. Comparison between SMM and isolated MRLC revealed that the phosphorylation rate of SMM is slower than that of isolated MRLC. To dissect the molecular mechanism responsible for this difference, we measured interactions between ZIPK and SMM by cosedimentation assay. The result suggests that the C-terminal domain of ZIPK interacts with the heavy chain of SMM, and as a result, competitive binding of ZIPK to MRLC and the myosin heavy chain suppresses phosphorylation of SMM compared to isolated MRLC. By incorporating the kinetic and dissociation constants obtained from mutant analysis and cosedimentation assays, respectively, a simple kinetic model reasonably well reproduced the time courses of phosphorylation for both isolated MRLC and SMM. This provides systemic insight into the regulatory mechanism of myosin contractility by ZIPK.

Repeat expansions in the genome are associated with numerous genetic diseases. The instability of repeat sequences is driven in part by slipped-out structures, such as hairpins. APOBEC3A (A3A), a cytosine deaminase, preferentially targets single-stranded DNA, including repeat regions capable of forming such secondary structures. In this study, we investigated how small molecules that selectively bind C–C mismatches in CCG hairpin repeats modulate A3A-mediated deamination. Using model oligonucleotides containing (CCG)₉ repeats and mismatch-binding ligands (MBLs: AmND and AmBzND), we show that these ligands selectively stabilize the stem regions of hairpin structures, suppressing deamination in the stem and directing A3A activity to the loop regions. The inhibitory effect was dose-dependent, and deamination occurred preferentially at loop cytosines. These findings demonstrate that hairpin stabilization with small molecules can modulate A3A site-selectivity with implications for understanding repeat instability and its therapeutic control. Furthermore, this approach may serve as a basis for developing chemical tools to manipulate repeat-associated genome functions.

Sodium ions are classically conceptualized as negative allosteric modulators for G protein-coupled receptors, although there have been reports of either positive allosteric modulation or no effect of sodium on GPCR function. Here, we identified opposing actions of sodium on μ and κ opioid receptors. We utilized a variety of methods including radioligand binding, real-time conformational monitoring of transitions using bioluminescence resonance energy transfer, and signaling assays using the TRUPATH resource. At the μ receptors, sodium behaved as a negative allosteric modulator of binding, conformational transitions, and signaling. Intriguingly, bitopic μ agonists displayed transducer-specific effects on conformational transitions and signaling sodium concentrations. By contrast, at the κ opioid receptor, sodium negatively modulated agonist binding and positively modulated conformational transitions and signaling. Taken together, these findings support the notion that the differential sensitivities to sodium concentrations will result in opposing effects on the cell surface and intracellular signaling.

Fusaric acid is a toxic metabolite produced by several Fusarium species, which causes wilt disease in many plants. Among other functions, fusaric acid allows the fungus to outcompete soil bacteria. Understanding mechanisms by which bacteria mitigate the toxic effects of fusaric acid is therefore of interest in terms of controlling Fusarium wilt. The soil bacterium Burkholderia thailandensis encodes a predicted fusaric acid-binding membrane transporter belonging to the FusC2 family. The fusC2 gene is annotated as part of an operon, also encoding an isochorismatase (gene named isoC) and a member of the multiple antibiotic resistance (MarR) family of transcription factors. Isochorismatase converts isochorismate to the antifungal compound 2,3-dihydroxybenzoate. We show here that the transcription factor, which we named FusR2, binds specifically to the promoter of the fusR2-isoC-fusC2 operon and that FusR2 induces marked changes in DNA conformation, as evidenced by hypersensitive DNase I cleavage sites. Fusaric acid induces the expression of fusR2-isoC-fusC2, and it binds directly to FusR2, as shown by thermal shift assays. While the presence of fusaric acid is compatible with DNA binding by FusR2, it eliminates the hypersensitive DNA cleavage. We propose that FusR2 imposes a DNA conformation, which adversely affects the ability of RNA polymerase to bind, whereas fusaric acid binding to FusR2 results in an altered DNA binding mode, in which the RNA polymerase can compete with FusR2 for DNA binding to initiate transcription. By this mechanism, fusaric acid induces the expression of genes encoding both an efflux pump and an enzyme involved in the production of an antifungal metabolite.

Avermitilol synthase from Streptomyces avermitilis (SaAS) is a high-fidelity class I terpene cyclase that converts farnesyl diphosphate into a highly strained, 6–6–3 tricyclic sesquiterpene alcohol. The mechanism of avermitilol formation proceeds through a 10–3 bicyclic intermediate, bicyclogermacrene, which undergoes proton-initiated anti-Markovnikov addition to two separate C═C bonds in a transannulation mechanism that forms the 6–6–3 tricyclic skeleton, with quenching by water to yield avermitilol. Small amounts of a side product, viridifloral, result from Markovnikov addition to one of the reactive C═C bonds. Here, we present enzymological studies of SaAS to establish the substrate scope and metal ion dependence for catalysis, and we present crystal structures of SaAS complexed with a variety of ligands that partially mimic carbocation intermediates in catalysis. Interestingly, these structures show that two water molecules remain trapped in a polar crevice in the active site regardless of the ligand bound. Structure–activity relationships for site-specific mutants yield key insight into the catalytic importance of these trapped water molecules. Specifically, T215 normally hydrogen bonds with water molecule W1, but the T215V substitution breaks this hydrogen bond and causes W1 to shift by 1.3 Å to form a hydrogen bond with W300. Avermitilol generation is completely lost in this mutant, but the generation of viridifloral and another side product is enhanced. We conclude that the T215V substitution causes water molecule W1 to align for reaction with the tertiary and not the secondary carbon in the reactive C═C bond of bicyclogermacrene.

The transcription of the HIV-1 long-terminal repeat (LTR), driven by the viral transactivator Tat, represents a distinct and druggable step in HIV-1 replication. Targeting Tat-mediated transcription is a promising antiretroviral strategy due to its mechanistic distinction from host cell transcription, although specificity remains a challenge due to potential off-target effects. To identify selective inhibitors, a two-step screening approach was employed. A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was first used to identify compounds that disrupt Tat–trans-activation response (TAR) RNA interactions, yielding 655 initial hits, including five 2-phenyl acrylate derivatives with >50% inhibition. A secondary library of 194 structurally related compounds was then screened using a dual-luciferase reporter assay to evaluate transcriptional inhibition and cytotoxicity. From this, 46 compounds met selection criteria (>50% F-Luc inhibition, > 90% R-Luc activity, > 90% viral inhibition, and >70% cell viability). Among them, methyl (E)-2-(2-((4-(decyloxy)phenoxy)methyl)phenyl)-3-methoxyacrylate showed the highest potency, with IC₅₀ and EC₅₀ values of 1.44 and 0.83 μM, respectively. Time-of-addition (TOA) assays indicated inhibition of the Tat-dependent transcription phase. Surface plasmon resonance analysis revealed binding to the Tat peptide but not to TAR RNA, suggesting the binding target for 019854-B06. Immunoblotting and coimmunoprecipitation showed that 019854-B06 neither promotes Tat degradation nor disrupts the Tat/CycT1 complex, supporting a Tat-centric, nondegradative mechanism. Therefore, this study identifies a novel 2-phenyl acrylate-based inhibitor of Tat-mediated HIV-1 transcription through an integrated biophysical and functional screening strategy.

Long COVID, or postacute sequelae of COVID-19 from SARS-CoV-2 infection, is a persistent debilitating disease affecting multiple systems and organs. Long COVID pathophysiology is a complex and not fully established process. One prevailing theory is that the formation of fibrin amyloid microclots (fibrinaloids), due to SARS-CoV-2 infection, can induce persistent inflammation and capillary blockage. An association between the amyloidogenic Spike protein of SARS-CoV-2 and impaired fibrinolysis was made when it was observed that fibrin clots formed in the presence of a mixture of amyloid fibrils from the spike protein mediated resistance to plasmin lysis. Here, we use purified components from the coagulation cascade to investigate the molecular processes of impaired fibrinolysis using seven amyloidogenic SARS-COV-2 Spike peptides. Five of seven Spike amyloid fibrils appeared not to substantially interfere with the fibrinogen–fibrin–fibrinolysis process in vitro, while two spike fibrils were active in different ways. Spike601 amyloid fibrils (sequence 601–620) impaired thrombin-mediated fibrin formation by binding and sequestering fibrinogen but did not affect fibrinolysis. On the contrary, fibrin clots formed in the presence of Spike685 amyloid fibrils (sequence 685–701) exhibited a marked resistance to plasmin-mediated fibrinolysis. We conclude that Spike685 amyloid fibrils can induce dense fibrin clot networks as well as incorporate fibrin into aggregated structures that resist fibrinolysis. Our study proposes a molecular mechanism for how the Spike protein of SARS-CoV-2 could contribute to the formation of fibrinolysis-resistant microclots observed in long COVID.

Hypoxia is a hallmark of the tumor microenvironment that profoundly alters the cellular metabolism and epigenetic regulation. In this study, we investigated how oxygen limitation reprograms histone methylation in glioblastoma cells by integrating stable isotope tracing with high-resolution proteomics and epigenomics. Using deuterium-labeled serine and the RQMID-MS platform, we demonstrated that hypoxia impairs methyl group transfer from serine to histones due to the downregulation of the vitamin B₁₂ transporter TCN2, which is critical for homocysteine remethylation and SAM synthesis. Despite this blockade in one-carbon metabolism, global histone methylation patterns were not uniformly suppressed. Instead, we observed site-specific changes driven by altered expression of methyltransferases and demethylases, particularly decreased KMT1F (H3K9 methylation) and KMT2B (H3K4 methylation) and increased KDM2A (H3K36 demethylation), KDM3A (H3K9 demethylation), and KMT5A/SETD8 (H4K20 monomethylation). These findings reveal that the histone methylation landscape under hypoxia is governed by a compensatory interplay between one-carbon metabolism and chromatin-modifying enzyme regulation.