Biochemistry Biochemistry最新文献

In the context of biohydrogen production, the O₂-stable Group B [FeFe]-hydrogenase from Thermosediminibacter oceani has attracted significant interest due to its distinctive TSCCCP motif near the active site, which contains an additional (third) cysteine residue that is absent in the TSCCP motif of standard Group A hydrogenases. The precise role of the additional cysteine residue in H₂ production has remained an open question. In this study, we sought to contribute to the understanding of this cysteine’s role in H₂ production by combining molecular dynamics (MD) simulations, site-directed mutagenesis, biochemical assays, and Fourier-transform infrared (FTIR) spectroscopy. Remarkably, a cysteine-to-serine exchange variant (TSSCCP) demonstrated enhanced H₂ production activity without compromising the O₂-stability of ToHydA, offering new insights into its functional dynamics.

Phytochromes are sensory photoreceptors in eukaryotes and prokaryotes that control physiological processes. In prototypical phytochromes, photoisomerization of the methine-bridged tetrapyrrole of the Pr state is the first step in (de)activating the photoreceptor. The underlying reaction sequence runs through a series of intermediate states. Among them, the Meta-Rc state plays a critical role since it precedes the formation of the Pfr state, which is linked to the functional secondary structure transition of the tongue, a phytochrome-specific peptide segment. In this work, we have studied the structure and reactions of Meta-Rc of the bacterial phytochrome Agp1 (Agrobacterium fabrum) by IR difference and resonance Raman spectroscopy. It is shown that the formation of Meta-Rc is associated with the enolization of the terminal ring D and the deprotonation of ring B or C, whereas reprotonation of the chromophore occurs with the decay of Meta-Rc. Proton migration represents the essential trigger for the secondary structure transition of the tongue since the β-sheet and α-helix structures can be interconverted by changing the pH. The pH-dependent conformational equilibrium is observed in Meta-Rc at 250 K and in Pfr at 290 K, albeit with different pK_A values. The results show that the secondary structure transition is induced by chromophore-linked proton transfer steps rather than by conformational relaxations of the chromophore itself. In view of previous findings on the proton dependence of the reverse process in bathy phytochromes, we conclude that intramolecular proton transfer is an indispensable prerequisite for the secondary structure transition in phytochromes in general.

Protein prenylation is a post-translational modification promoting membrane association where isoprenoid lipids attach to C-terminal cysteines of eukaryotic proteins such as Ras and Rho GTPases, nucleus lamins, and G-protein subunits. Three enzymes catalyze this process: farnesyltransferase (FTase) and geranylgeranyltransferase type I and II (GGTase I and RabGGTase). FTase and GGTase-I recognize C-terminal CaaX motifs, of which the terminal amino acid confers specificity. Due to its involvement in oncogenic Ras activation, FTase has become a major anticancer target for drug development. Although first-generation FTase inhibitors failed in clinical trials in many cancers due to compensatory geranylgeranylation of KRAS and NRAS, they remain effective against HRAS-driven tumors and other pathologies, such as Hutchinson–Gilford progeria syndrome. The FTase inhibitor A-176120 was reported to compete with farnesyl and not KRAS. However, our crystallographic and biochemical analyses reveal that A-176120 sterically interferes with the engagement of the KRAS CAAX motif, reducing, but not abolishing, its binding to FTase.

Protein degradation through the autophagy–lysosome process by eukaryotic cells is a major pathway to remove unwanted proteins, organelles, and invading pathogens. It is also an emerging intervention strategy to selectively eliminate inaccessible toxic amyloid proteins to prevent amyloid β (Aβ)-induced neurotoxicity. Currently, there is no natural product-derived peptide that targets amyloid proteins for degradation through the autophagy–lysosome pathway. We recently discovered a new peptide family from Ginkgo biloba nuts, which we termed β-ginkgotides. The prototype β-gB1 is 20-residue in length, cross-braced by three disulfides, and stable to proteolytic degradation. Importantly, it has an LC3-interacting region (LIR) motif, which promotes selective autophagy to degrade harmful proteins and to prevent cell death. Here, we show that β-gB1 is cell-penetrating, primarily entering cells through energy-dependent endocytosis, and protects Aβ-induced neurotoxicity using an SH-SY5Y neuronal cell-based model. Functional studies using synthetic β-gB1 revealed that it impedes Aβ accumulation and reverses the altered gene expression associated with Alzheimer’s disease (AD) pathophysiology induced by Aβ. Importantly, β-gB1 maintains cellular homeostasis and enhances the clearance of Aβ aggregates through selective autophagy, thereby safeguarding neurons from Aβ toxicity. Collectively, these results support that β-ginkgotide is a first-in-class cysteine-rich peptide (CRP)-based targeted protein degrader and underscore its potential as a novel and promising neuroprotective therapeutic to manage Aβ-induced neurotoxicity in AD and other neurodegenerative disorders.

Anti-CRISPR (Acr) proteins have long exemplified the viral counterattack against CRISPR-Cas immunity. By contrast, comparatively little is known about host proteins that may increase Cas effector activity. Recent work on a compact type V nuclease, Cas12p, demonstrates that this phage-associated effector depends on the bacterial thioredoxin TrxA for efficient DNA cleavage. TrxA binds a dedicated thioredoxin-binding (TB) domain on Cas12p through a redox-sensitive interaction, promoting an active conformation competent for DNA cleavage. This finding adds to a small but growing set of CRISPR activators and highlights that CRISPR-Cas systems are not static defense modules but dynamic networks shaped by auxiliary factors that can fine-tune their activity.

Checkpoint inhibitors targeting the PD-1/PD-L1 axis are key immunotherapies, but the dynamic and flexible nature of PD-1 complicates rational antibody engineering. Here, we use computational saturation mutagenesis, AlphaFold prediction, and molecular dynamics (MD) simulations to evolve pembrolizumab variants with suitable binding. Seven engineered antibodies form additional salt bridges and hydrophobic contacts via refolding of both the antibody and the PD-1 interface. One variant, m7p.5, displays improved biphasic kinetics and high-affinity binding (K_D,apparent = 62 pM). Structural changes include an α-helix to loop transition in the antibody heavy chain and a 4.6-Å Cα shift of a PD-1 loop. These results show that computational evolution can access binding modes inaccessible to traditional rigid structural design, enabling high-affinity antibodies for flexible targets. It is demonstrated that our integrated computational approaches including MD simulations can generate new picomolar high-affinity antibodies targeting specific epitopes of proteins that may be intrinsically flexible and are difficult to target with reasonable computational cost, which would be far less than an experimental cost for finding new antibodies with equivalent binding affinities. This study provides a new tool that can be combined with other artificial-intelligence-based antibody generation against PD-1 from the existing anti-PD-1 antibody library with broad applications in protein–protein interactions.

The strong, polar covalent nature of C–F bonds contributes to the permanent nature of per- and polyfluoroalkyl substances (PFAS). PFAS are toxic to humans. Here, we have examined the ability of the small, globular milk protein β-lactoglobulin to bind PFAS. This protein transports hydrophobic and amphiphilic compounds, including retinol and fatty acids, for vision and brain development; therefore, understanding its interactions with PFAS is significant. The crystal structures of β-lactoglobulin complexed with PFOA (perfluorooctanoic acid) at 2.0 Å, PFOS (perfluorooctanesulfonic acid) at 2.5 Å, and PFDA (perfluorodecanoic acid) at 2.0 Å reveal the high affinity of the compounds for the central calyx of β-lactoglobulin, which is the canonical retinol and fatty acid binding site. Analyses of the data indicate significant hydrophobic interactions stabilizing the binding of the PFAS hydrophobic “tails” within the calyx and interactions between Lys60 and Lys69 and the PFAS polar head groups. Comparative structural analysis revealed the presence of an open conformation of the EF loop containing the Glu89 latch residue in the complexed structures compared to the apo-form. Molecular dynamics (MD) simulations revealed the high stability of PFAS binding and the attainment of energy minima in all complexes. The average binding energy of PFDA in the β-lactoglobulin calyx was −25 kcal/mol, which was higher than that of PFOS (−21 kcal/mol) and PFOA (−23 kcal/mol) due to the increased van der Waals interactions between the longer hydrophobic chain of PFDA and β-lactoglobulin. This work advances a mechanism by which β-lactoglobulin can recruit PFAS and act as a transporter for the “forever” chemical, potentially mediating its neurotoxicity.

Yin-Yang 1 (YY1) is a CCHH-type classical zinc finger (ZF) protein that plays diverse roles in gene expression, acting as both a transcriptional activator and repressor, which is important for DNA repair, neuronal development, and oncogenesis. Classical ZFs adopt a ββα fold upon Zn(II) binding, and YY1 contains four CCHH-type domains. The two central domains (ZF2 and ZF3) are known to directly bind to DNA. Although ZFs have traditionally been viewed as just structural domains, emerging data shows that ZFs can be modified by the gaseous signaling molecule hydrogen sulfide, H₂S, to form persulfides. These data are principally from proteomics studies from which several classical ZFs, including YY1, were identified as persulfidated. Herein, we report how the classical ZF YY1 is persulfidated by H₂S and the effects of persulfidation on DNA binding using three ZF constructs containing the second domain (YY1-ZF2), third domain (YY1-ZF3), and both the second and third domains (YY1-ZF2-ZF3). Persulfidation of all three constructs was observed using an NBF-Cl/dimedone tag-switch method. Persulfidation required Zn(II) and O₂. Superoxide, as measured by hydroethidine and superoxide dismutase experiments, was also observed as an intermediate. YY1-ZF2-ZF3 was also shown to bind to the adeno-associated virus P5 initiator and IL-6 promoter DNA via a fluorescence anisotropy assay. This ZF/DNA binding was abrogated by H₂S; however, when DNA was bound to YY1-ZF2-ZF3, it was unreactive to H₂S modification suggesting a protective effect of the DNA macromolecule. In addition, H₂S disrupted the secondary structure of all three YY1 constructs as measured by circular dichroism.

Modulation of the nervous system by gut microbiota through metabolic pathways is a key mechanism of communication within the gut–brain axis. A critical factor determining whether gut microbial metabolites can exert functional effects in the brain is their ability to cross the blood–brain barrier (BBB). However, current methods for assessing BBB permeability lack systematic, standardized approaches and advanced predictive technologies. Traditional experimental techniques are often costly and time-consuming compared to computational methods. To address these limitations, we developed an automated molecular simulation workflow to generate a high-quality data set of gut microbial metabolites annotated with thermodynamic features related to BBB permeability. Based on this data set, we constructed an interpretable thermodynamic evaluation framework capable of accurately identifying key factors that influence transmembrane transport. The robustness and predictive power of our models were validated using two authoritative benchmark data sets, confirming their ability to reliably distinguish BBB-permeable from nonpermeable compounds. Furthermore, our findings highlight the substantial potential of gut microbiota metabolism to influence BBB permeability via metabolic pathways. Overall, this study provides a powerful tool for identifying gut microbiota-derived metabolites with potential biological activity in the brain and introduces a novel paradigm for the intelligent prediction of BBB permeability.

Thousands of recently discovered microproteins represent a new frontier in the search for functional and disease-causing genes. Though shorter than canonical proteins, some microproteins contain signal peptides and are predicted to produce secreted peptides. However, whether any of the microprotein-derived secreted peptides possess biological activity remains underexplored. Here, we screen a small library of secreted peptides from the microproteome by measuring signaling downstream from GPCRs. This approach identified several cAMP-stimulating peptides, including a secreted peptide from a “non-coding” HLA complex P5 RNA (HCP5). The HCP5-secreted peptide (HCP5-SP) is encoded by a small open reading frame embedded in the HCP5 mRNA. In vitro assays with synthetic HCP5-SP and HCP5-SP analogs validated its cAMP-stimulating activity and revealed the necessity for the wild-type C-terminal sequence for activity. Furthermore, HCP5-SP promotes the proliferation of HEK293T cells, providing an alternative mechanism that might explain some of the cancer biology associated with HCP5 mRNA. In summary, this work establishes a workflow for the preliminary identification of bioactive microproteins and demonstrates that the vast, largely untapped microproteome is a source of novel bioactive endogenous peptides.