Biochemistry Biochemistry最新文献

The endoplasmic reticulum (ER), the largest cellular organelle, is crucially dependent on its redox organization. First, to optimize disulfide bond formation in nascent proteins, it maintains a relatively oxidizing environment, reminiscent of the extracellular space. Second, it harbors several oxidoreductases from the protein disulfide isomerase (PDI) family, together with Ero1α oxidase and chaperones, which compose interplaying oxidative, reductive, and chaperone pathways to optimize protein processing. Third, disulfide formation and reshuffling in client proteins, involving thiol oxidation and disulfide exchange reactions, connect proteostasis to ER/cellular redox homeostasis. ER redox folding involves Ca²⁺-dependent liquid phase separation of PDI complexes. Calcium fluxes heavily interplay with dynamic redox regulation. ER stress disrupts the ER redox state and, in turn, is also regulated by cellular redox processes. Moreover, the ER makes membrane contacts with many other organelles such as plasma membrane, peroxisomes, and mitochondria, which are hubs for mutually dependent oxidant and calcium-linked effects. Furthermore, the ER redoxome extends to other subcellular and extracellular locations, a process we termed the “ER-dependent outreach redoxome (ERDOR)”. ERDOR can occur by overflow of ER products such as H₂O₂, mobility of ER-associated domains or, mainly, via ER oxidoreductase translocation. The ER establishes a particular communication with the extracellular milieu via translocation of PDIs. Despite the low levels of extracellularly located ER oxidoreductases, they redox-regulate several molecular targets and may compose a peri/epicellular redox network. This article provides a comprehensive overview of the ER redoxome as an important emerging frontier to understand not only redox proteostasis but also intra- and intercellular redox communication.

Several bacterial pathogens secrete multidomain enzymes known as lytic polysaccharide monooxygenases (LPMOs) that are important for virulence. One example is the Vibrio cholerae virulence factor GbpA (VcGbpA), in which an N-terminal LPMO domain is followed by two domains of unknown function called GbpA2 and GbpA3, and a C-terminal chitin-binding domain called CBM73. In-depth functional characterization of full-length and truncated variants of VcGbpA and a homologue from V. campbellii (previously V. harveyi, VhGbpA) showed that the catalytic LPMO domains of these proteins exhibit properties similar to natural single-domain LPMOs with established roles in chitin degradation. Interestingly, binding to chitin and efficient degradation of this substrate were affected by the presence of the GbpA2 and GbpA3 domains. Combined with structural predictions and analyses of sequence conservation, our data show that GbpA3 has evolved to interact with the reduced catalytic copper site in the LPMO domain to prevent off-pathway reactions in the absence of substrate. Substrate binding by CBM73 weakens this interaction, enabling the activation of the LPMO only when substrate is present. These observations shed new light into the functionality of these multidomain LPMOs and uncover a novel mechanism for regulating LPMO activity.

The enhanced catalytic activity (superactivity) of iron-depleted apo-human serum transferrin (apo-hTF) in the presence of cationic surfactants with varying chain lengths has been investigated in this work. The progress of ester hydrolysis of two different esterase substrates, para-nitrophenylacetate (PNPA) and 4-methylumbelliferylacetate (4-MUA), was monitored spectroscopically. Catalytic activity of apo-hTF gets enhanced with increasing concentrations of cationic surfactants, up to the micellar concentration, followed by a gradual decrease at postmicellar concentrations. However, the catalytic performance of the protein remained silent in its native form, in the presence of anionic and neutral surfactants, guanidinium hydrochloride-denatured conformation, temperature-induced aggregated form, and liquid–liquid phase-separated (LLPS) form of the protein. This work sheds light on the importance of the location and alignment of amino acids in the catalytic hub and the approachability of the substrate at the active site in micellar catalysis systems. These results provide new insights into enzyme–substrate interactions in the domain of micellar catalysis, potentially aiding the design of surfactant-based catalytic systems.

DNA single-strand breaks (SSBs) containing covalent DNA–protein cross-links at 5′-termini (5′-DPCs) are produced from the C1′-oxidized abasic site, 2-deoxyribonolactone. These adducts need to be removed for SSB repair because 5′-phosphate is required for strand ligation. Prior studies showed that 5′-DPCs can undergo proteolysis by the 26S proteasome. However, how the remaining 5′-DNA-peptide cross-links (5′-DpCs) are removed is unclear. Herein, we found that a chemically synthesized and site-specific 5′-DpC can be repaired by HeLa cell nuclear extracts, and human flap-endonuclease 1 (hFEN1) plays an essential role in the DpC excision. We also synthesized a model 5′-DPC by reductive amination and showed that prior proteolysis of the cross-linked protein by trypsin greatly facilitated the DPC repair in HeLa cell nuclear extracts. Our findings suggest that 5′-DPCs within SSBs can be repaired by proteolysis followed by the long-patch base excision repair pathway.

Single-stranded (ss) DNA binding protein (gp32) serves as the central regulatory component of the multisubunit T4 bacteriophage DNA replication system by coordinating the system’s three functional subassemblies, resulting in phage DNA synthesis in T4-infected Escherichia coli cells at the high speeds (∼1000 nts s^–1) and the high fidelity (<1 error per 10⁷ nts) required for genomic function within this cellular ecosystem. Gp32 proteins continuously bind to, slide on as cooperatively linked clusters, and unbind from transiently exposed single strands of DNA to carry out their coordinating functions. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within gp32 clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule Förster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine single strands of varying strand length and polarity near model ss-dsDNA [3′,5′-oligo-(dT)_14,15-dsDNA] junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by strand polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTDs might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32 nucleoprotein filaments involved in phage DNA replication and related processes.

The receptor binding domain of hemagglutinin (HA) of influenza viruses contains three key regions for binding its endogenous carbohydrate receptors: loop-130, helix-190, and loop-220. To effectively predict the binding of HA with endogenous glycan ligands or designed inhibitors, the present study proposed a hypothesis that these ligands need to form stable interactions with at least two of the three critical regions simultaneously in the binding site. The testing of the hypothesis employed multiple HA variants, including H1, H3, H7, H17, and H18, with both α-2,6 and α-2,3-linked sialosides. Observations from molecular dynamics simulations are consistent with the experimentally discovered binding preferences for HA. To extend the proposed hypothesis to the antiviral drug design, it was further tested by using a noncarbohydrate receptor that formed a cocrystal complex with H5, N-cyclohexyltaurine (NCT), and an experimentally measured inhibitor, curcumin. Observations from the molecular models for these structurally distinctive molecules provided further test for the hypothesis and extended the applicability to noncarbohydrate ligands. The proposed hypothesis provided an alternative explanation for the binding preference of HA proteins, a fast approach to determine the binding stability of a ligand, and insights into the design of antiviral drug molecules targeting HA.

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.