Biochemistry Biochemistry最新文献

UBQLN2 is a member of the UBL-UBA domain protein family that functions as extrinsic substrate receptors for the 26S proteasome. UBQLN2 has been shown to undergo phase separation in vitro. In cells, UBQLN2 forms condensates that may be of importance for tuning protein degradation via the ubiquitin-proteasome system and potentially of relevance for UBQLN2-linked amyotrophic lateral sclerosis (ALS). Here we show that UBQLN2 is ubiquitylated on lysine residues in the N-terminal UBL domain. The C-terminal region of UBQLN2 is lysine-depleted, and we show that introducing lysine residues in this region leads to its E6AP-dependent degradation. The UBL domain critically stabilizes UBQLN2 and protects it from proteasomal degradation. Fusion of ubiquitin to the UBQLN2 N-terminus stabilizes UBQLN2 and increases its propensity for locating in puncta, indicating that ubiquitylation of the UBQLN2 UBL domain regulates abundance and localization.

Zika virus infections in humans were identified in several African countries in the 1950s and spread globally in the mid-2010s. Cases began to surge in South America around 2016, coinciding with a rise in severe developmental disorders in babies born to infected mothers. Gaining a deeper understanding of ways to treat infection is crucial. To this end, we sought to investigate the dynamics of Zika Virus Protease NS2B-NS3, a drug target that will enable effective treatment of infection and may be invaluable in treating more lethal variants that may eventually emerge. Specifically, we employed hydrogen–deuterium exchange mass spectrometry on NS2B-NS3 to observe which regions of the protein are quick or slow to exchange, and how these exchange patterns change in the presence of an allosteric inhibitor. From these studies, we observe that Zika Virus Protease populates the open conformation when it is unbound or bound to the allosteric inhibitor MH1. We further identified a single substitution in NS3, A125C, that directly blocks allosteric inhibition. The observed deuterium uptake patterns provide a detailed view of Zika Virus Protease dynamics in unbound and inhibitor-bound states, allowing us to visualize how allosteric binding at NS3 prevents closure of NS2B and propagates structural perturbations that together result in protease inhibition. Importantly, our studies predict that interactions between the NS2B cofactor and the NS3 core determine the potency of this class of inhibitors across the flaviviral proteases. Pan-flaviviral inhibitors would provide invaluable antiviral modalities, and insights from these studies should aid in their development.

Methylglyoxal (MGO) and glyoxal (GO) are reactive carbonyl species (RCS) generated as side products in glycolysis and carbohydrate, protein, and fat catabolism, which are enriched in most cancer cells. MGO/GO-induced nonenzymatic glycation on histones plays pathophysiologically important roles in regulating the three-dimensional architecture of cellular chromatin and cancer development. In our previous studies, we have uncovered that enzymes DJ-1 and PAD4 exhibit “glyoxalase” and “deglycase” activities to antagonize the MGO/GO-modifications of histones. We also found that the general inhibition of histone deacetylases using suberoylanilide hydroxamic acid (SAHA) antagonized histone MGO-glycation due to the direct competition of reactive sites (i.e., lysine residues). Here, we report that a histone deacetylase, sirtuin 2 (SIRT2), functions as a “semi-deglycase” that removes lactic and glycolic acids from ε-N-l-lactyllysine and hydroxyacetyllysine residues, which are derived from MGO/GO-lysine adducts through the isomerization catalyzed by DJ-1. Overall, SIRT2 is a newly identified regulator for histone glycation, which can prevent the cytotoxicity of MGO and GO by eventually converting them into lactate and glycolate with the assistance of an enzymatically inactive DJ-1 mutant (i.e., DJ-1-C106A).

Integrin α4β1 plays a central role in immune cell adhesion and trafficking and contributes to the adhesion of mesenchymal cells, making it a valuable target for designing cell-adhesive biomaterials. Peptide ligands based on the Leu-Asp-Val (LDV) motif of fibronectin have been widely studied, yet their minimal active sequences and potential as cell-adhesive molecules remain insufficiently characterized. Here, we systematically evaluated a panel of LDV-related peptides for their ability to promote α4β1-mediated adhesion of Jurkat T cells. Among six candidates, EILDVPST, derived from the CS1 domain of fibronectin, exhibited the strongest adhesion activity. Truncation and alanine-scanning analyses identified EILDVPS as the minimal sequence with full activity, with Leu and Asp serving as the critical binding core and surrounding residues providing structural support. The cyclization of EILDVPS markedly enhanced its adhesion-promoting activity, providing direct evidence that conformational constraint increases α4β1-binding affinity. Comparative assays with the high-affinity peptidomimetic ligands BIO1211-C and LLP2A-C demonstrated that although cEILDVPS was less potent, it supported long-term adhesion culture of T cells, comparable to that achieved with these ligands. Together, these findings establish EILDVPS as a minimal α4β1-binding motif, demonstrate the utility of cyclization for enhancing activity, and highlight the potential of LDV-based peptides for the adhesion culture of α4β1-expressing cells and development of integrin-targeted biomaterials.

The reduction of cytochrome P450 (P450s) is a prerequisite for almost all P450 reactions, where electrons are shuttled to the active site heme from protein electron-transfer (redox) partners. However, this process is more complicated than simple electron transfer due to complex protein–protein interfacial interactions. To improve our understanding of redox partner complexation and selectivity, we investigated ferredoxin interactions with a bacterial P450, CYP108A1 (P450terp), a homologue of the model system, P450cam. We recently solved the crystal structure of the P450terp–substrate complex, and here, we report the crystal structure of terpredoxin (Tdx), the native redox partner of P450terp. Utilizing protein–protein docking and molecular dynamics simulations, we probed possible P450terp–Tdx complexes. Not surprisingly, the surfaces that interact in the P450terp–Tdx complex are the same as in the P450cam–Pdx complex, although the specific interactions are substantially different. Glu38 (E38) in Tdx was predicted to form an ion pair with Arg114 in P450terp, similar to the D38-R112 ion pair in the P450cam–Pdx complex. We therefore prepared an E38L variant of Tdx that lowered but did not eliminate activity. Unlike P450cam, P450terp can be supported by foreign redox partners, including Pdx. Mutations of Pdx designed to mimic Tdx were unable to appreciably increase the turnover rate. To investigate whether redox partner binding could influence structural changes in P450terp, we measured the change in the stability of the P450 oxycomplex in the presence of the ferredoxins with the highest turnover rates and found a correlation between the ferredoxins that enabled turnover and the stability of the oxycomplex.

Dynamic protein loops can act as molecular gates that stabilize enzyme–substrate complexes, yet the underlying motions are poorly defined. Here, we dissect the role of loop 3 in an NADH:quinone oxidoreductase (NQO, UniProt Q9I4V0) from Pseudomonas aeruginosa PA01 in governing substrate binding and catalysis. Previous mechanistic and structural studies proposed that loop 3 fluctuations regulate substrate binding; however, an associated atomic-level understanding of the conformational changes is lacking. We probe the role of loop 3 dynamics in substrate capture and catalysis by mutating conserved P78 to glycine, which perturbs the gate rigidity. Steady-state kinetics with NQO-P78G and NQO-WT at varying concentrations of NADH and coenzyme Q₀ established a 3.5-fold decrease in the K_CoQ0 value, a 2.0-fold reduction in the k_cat value, and a 1.8-fold increase in the k_cat/K_CoQ0 value. The anaerobic reductive half-reaction of NQO-P78G with NADH yielded a ≤3.5-fold decrease in the k_red value and an estimated 80-fold increase in the K_d value compared to NQO-WT. Molecular dynamics simulations of ligand-free NQO-P78G and NQO-WT suggest that the P78G mutation disrupts interdomain interactions, allowing loop 3 to sample more open conformations. The combination of mechanistic and computational experiments suggests that more open gate conformations minimally promote access of the smaller coenzyme Q₀ substrate to the active site. In contrast, the bulkier NADH substrate is less likely to associate, as the more open conformations prevent key interactions with NQO gate residues from forming. These results build on previous studies with NQO by demonstrating that altering loop 3 gate rigidity modulates substrate binding.

W1282X CFTR is the most prevalent CF-causing variant among cystic fibrosis patients of Ashkenazi descent and a mutational defect for which targeted drug therapy is not available. We show that administration of the potentiator VX-770 can augment levels of truncated W1282X CFTR in the plasma membrane, demonstrating that an established gating activator (i.e., “potentiator”) also rescues W1282X protein expression and surface localization (i.e., “corrector” function). Additionally, acute in vitro treatments with approved modulators VX-809 or VX-661 result in immediate potentiation of W1282X-dependent ion transport, showing that F508del CFTR correctors also augment W1282X CFTR channel activity. To investigate the mechanism, we tested a CFTR variant (G551D) exhibiting higher levels of CFTR-dependent potentiation following corrector treatment. Clinically approved CFTR correctors VX-445, VX-121, and VX-809 elicited potentiation of G551D CFTR. Forskolin dose dependence and molecular dynamic simulations indicated that corrector molecules promote acute CFTR gating by modifying protein conformation and enhancing heterodimerization of nucleotide binding domains, leading to potentiator-like effects. Although W1282X is poorly responsive to “readthrough” agents such as G418, the drug unexpectedly increases W1282X mRNA, augments surface-localized (truncated) protein, and promotes CFTR function, even in the absence of detectable stop codon suppression. Moreover, unlike other CFTR mutations such as F508del, proteasome blockade using ALLN partially rescues W1282X at the plasma membrane. These results highlight ways in which detailed mechanistic analysis and modulator profiling are needed to characterize CFTR mutations such as W1282X and that modulator function in rare variants can be quite distinct from classical findings based strictly upon F508del CFTR.

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.