Biochemistry Biochemistry最新文献

Supramolecular β-sheet peptide nanomaterials are of critical interest due to their relevance in amyloid disorders and are increasingly valued for applications in regenerative medicine, tissue engineering, and antimicrobial design. Amphipathic peptides, particularly those with alternating hydrophobic and hydrophilic residues, readily form amyloid-like pleated β-sheet fibrils. It has been demonstrated that the amino acid sequence order of isomeric peptides dramatically influences the self-assembly propensity of the resulting sequences as well as the morphology of the assembled pleated β-sheet nanomaterials. This was substantiated by our previous investigations of the peptides Ac-(FKFE)₂-NH₂ (L1), Ac-(FK)₂(FE)₂-NH₂ (L2), Ac-KE(F)₄KE-NH2 (L3), Ac-(KFFE)₂-NH₂ (L4), and Ac-FF(KE)₂FF-NH₂ (L5). Recently, interest in the Pauling and Corey rippled β-sheet motif, composed of coassembled enantiomeric l- and d-peptides in which the l- and d-enantiomers are organized in an alternating fashion, has been revitalized, although understanding of the rippled β-sheet fold lags far behind that of the naturally occurring pleated β-sheet. Herein, we interrogate the scope of rippled β-sheet formation by extending our previous study of the L1-L5 peptides to enantiomeric mixtures of these sequences to understand the effect of sequence order on rippled β-sheet formation. These integrated experimental and computational studies confirm that enantiomeric mixtures of these peptides have a significantly higher propensity to coassemble into putative rippled β-sheets than single enantiomers have to self-assemble into pleated β-sheets under the same solvent and concentration conditions. These findings extend our understanding of the rippled β-sheet motif and highlight the potential to exploit stereochemically diverse peptides in the design of next-generation biomaterials.

Obesity and its associated metabolic syndrome present significant therapeutic challenges, with current pharmacological interventions often falling short of replicating the multifaceted benefits of bariatric surgery. Recent advances in incretin-based therapies, particularly GLP-1 and GIP coagonists, have demonstrated substantial improvements in glycemic control and weight management, yet residual cardiovascular risk and lipid abnormalities persist. Fibroblast growth factor 21 (FGF21) has emerged as a promising protein to complement incretin pharmacology due to its potent lipid-lowering effects and favorable safety profile. This study describes the engineering of a balanced, long-acting triple agonist that simultaneously targets FGF21, GLP-1, and GIP receptors. Through strategic N- and C-terminal modifications and lipid conjugation, a novel optimized FGF21 analogue is engineered to exhibit enhanced potency, stability, and sustained pharmacokinetics compared to native protein. In Diet-Induced Obesity (DIO) mice, this FGF21 analogue achieves near normalization of body weight, superior to benchmark GLP-1 agonists, and demonstrates additive efficacy when combined with a GLP-1/GIP receptor coagonist. Based on this additivity, a unimolecular triagonist is engineered, and mechanistic studies confirm balanced receptor activity at the FGF21 and incretin receptors to achieve combinational pharmacology, with significant reductions in body fat, improved glucose tolerance, and extended duration of action. These findings position the FGF21/GLP-1/GIP triagonist as a first-in-class candidate for next-generation metabolic disease therapy, potentially approximating the efficacy of surgical intervention while addressing lipid disorders inadequately managed by current incretin therapies.

Nonpathogenic cellular prion protein (PrP^C) is expressed by neurons and other cells, regulating neurite outgrowth, cell survival, myelin maintenance, and immunity, yet the PrP^C-protein interaction network and signaling pathways that underlie PrP^C function remain incompletely understood. PrP^C is glycophosphatidylinositol-anchored in lipid rafts and reportedly interacts with membrane-bound proteins at the cell surface, including the N-methyl-d-aspartate receptor (NMDA-R), triggering cell-signaling responses. PrP^C may also be glycosylphosphatidylinositol (GPI)-anchored in extracellular vesicles or released from cells by proteases to interact with plasma membrane proteins in target cells. To identify PrP^C binding sites for the NMDA-R in an unbiased manner, we generated extracts from HEK293T cells transfected with the GluN1 and GluN2B NMDA-R subunits and performed a targeted series of co-immunoprecipitation experiments, peptide arrays, and protein structure analyses. We identified two sites in PrP^C that bind to the NMDA-R. One site was located in the N-terminal disordered region of PrP^C. This site is in a lysine-rich segment that incorporates the sequence previously identified as the biologically active PrP^C-derived peptide, P3. The second site was located in the C-terminal structured region of PrP^C within the α1 helix and β1 strand. PrP^C bound GluN1-GluN2B complexes as well as GluN1 in isolation. Notably, the N-linked glycans in PrP^C inhibited binding to GluN1. Mutation of PrP^C to incorporate a third glycosylation site further inhibited binding to GluN1. These results demonstrate binding sites in PrP^C that may mediate interaction with the NMDA-R when PrP^C is membrane-anchored to the cell of origin, released in extracellular vesicles, or shed from the cell surface by proteases.

The L-type voltage-gated Ca²⁺ channel (Ca_V1.2) controls gene expression, cardiac function, and neuronal excitability. Mutations in Ca_V1.2 that disrupt channel function are implicated in cardiac arrhythmias, vascular dysfunction, Timothy Syndrome, and epilepsy. Calcium-binding protein 1 (CaBP1) binds to the IQ-motif in Ca_V1.2 (residues 1640-1665), blocks Ca²⁺-dependent inactivation (CDI), and promotes Ca²⁺-dependent facilitation (CDF). CaBP1 is 56% identical in sequence to calmodulin (CaM), and both proteins bind competitively to the IQ-motif. Our binding studies reveal that Ca²⁺ binding to CaBP1 is enhanced more than 40-fold when CaBP1 is bound to the IQ peptide. Also, the IQ peptide binds to Ca²⁺-bound CaBP1 (dissociation constant of 45 ± 10 nM) with 100-fold higher affinity than IQ binding to Ca²⁺-free CaBP1. We present NMR structures of Ca²⁺-CaBP1 bound to the IQ peptide, which reveal CaBP1 residues (A107, F111, M128, L131, I144, and M165) that contact IQ residues (I1654, Y1657, and F1658). Also, IQ residue K1662 forms a salt bridge with CaBP1 residue D140, which may explain why a K1662 charge reversal mutation causes 4-fold weaker IQ binding to CaBP1. Electrophysiology studies suggest that CaBP1 acts to increase the Ca_V1.2 channel open probability (Po). We propose that Ca²⁺ binding to the third and fourth EF-hands of CaBP1 and the binding of Ca²⁺-bound CaBP1 to the IQ-motif are important for Ca_V1.2 channel activation.

Endonuclease G (EndoG) is a conserved endonuclease implicated in mitochondrial DNA (mtDNA) replication, maintenance of mtDNA integrity under oxidative stress, and the removal of nuclear and paternal mtDNA during apoptosis and early embryogenesis. Despite its biological significance, the substrates targeted by EndoG and its cleavage preferences remain unclear. Here, we characterize human EndoG (hEndoG) across diverse nucleic acid substrates, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), nicked and gapped dsDNA, modified dsDNA containing 8-oxoguanine (oxoG-DNA) and hydroxymethylated cytosine (5hmC-DNA), single-stranded RNA (ssRNA), and RNA/DNA hybrids. We show that hEndoG binds most of these substrates with only modest differences in affinity (∼10-fold), yet displays a particularly strong preference for cleaving oxidatively damaged DNA, including nicked and gapped dsDNA, and oxoG-DNA. Notably, hEndoG preferentially cleaves the strand opposite the gapped or nicked site, and it targets the complementary strand to the modified base in oxoG-DNA and 5hmC-DNA. Our structural modeling of hEndoG bound to ssDNA and dsDNA indicates that ssDNA is a favored substrate because its flexibility allows kinked conformations that position the scissile phosphate near the catalytic Mg²⁺ in the His-Me finger motif. Together, these findings support a critical role for hEndoG in preserving mitochondrial genome integrity under conditions of oxidative stress by selectively targeting and removing oxidatively damaged DNA.

Recently, a growing number of novel types of regulated cell death have been reported, including pyroptosis, necroptosis, and ferroptosis, among others. These types of cell death play crucial roles in a wide array of physiological functions such as metabolism, tissue injury and repair, chronic disease progression, and immune protection. However, specifically targeting cell death pathways for therapeutic purposes remains a challenge due to the unresolved complexities in pharmacological intervention. Nuclear receptor superfamily members, a class of prominent targets in drug discovery, are involved in diverse physiological and pathological processes. Investigating the regulatory functions between nuclear receptors and cell death is essential for understanding their roles in cell death and developing novel treatment methods for cell death-related diseases. This review discusses the mechanisms and functional significance of nuclear receptors in cell death across various physiological and pathological conditions, summarizes current ligands and compounds that facilitate targeting nuclear receptors to modulate cell death, and aims to promote the development of novel pharmacological strategies.

O-GlcNAcylation is a dynamic posttranslational modification regulated by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). It involves the attachment of N-acetylglucosamine to serine or threonine residues of proteins in the cytosol, nucleus, and mitochondria. As a dynamic and abundant modification, O-GlcNAcylation functions as a sensor of the cell's metabolic state. Fluctuations in O-GlcNAc levels of the adenosine (O-GlcNAc) signal cellular stress or metabolic changes and have been implicated in various human diseases. The overall impact of this modification is protein-dependent, underscoring the importance of studying its biochemical consequences in a protein- and site-specific manner. To achieve this, enzymatic and chemical strategies have been developed to incorporate O-GlcNAc into peptides and proteins. These synthetic glycopeptides and glycoproteins have been instrumental in elucidating how O-GlcNAcylation influences protein structure, function, and diverse biochemical pathways. Recently, the O-GlcNAcylation has also emerged as a tool for glycosylation-assisted folding of proteins and as a solubility tag for the chemical synthesis of glycopeptides and proteins. Here, we overview the current methods enabling the preparation of specific O-GlcNAc-modified proteins and highlight recent developments.

ISGylation is a ubiquitin-like post-translational modification that plays a central role in innate immune signaling. Conjugation of interferon-stimulated gene 15 (ISG15) to target proteins is initiated by the E1 enzyme Uba7, transferred to the E2 enzyme UbcH8, and completed by an E3 ligase. Specificity in this cascade is mediated by the ubiquitin-fold domain (UFD) of Uba7, yet the structural and mechanistic basis of E1-E2 recognition remains poorly defined. Here, we present the solution NMR structure and functional characterization of a human Uba7-UFD. NMR chemical shift perturbation experiments combined with site-directed mutagenesis delineate the UbcH8 interaction surface and identify residues critical for E1-E2 binding. The Uba7-UFD adopts a conserved ubiquitin-fold architecture but exhibits conformational flexibility in the unbound state. ¹⁵N relaxation measurements show a globally well-folded domain with localized ps-ns time scale dynamics within the β2/β4 E2 binding surface and the acidic loop spanning residues 996-1008. Upon UbcH8 binding, relaxation parameters shift toward those expected for a larger effective molecular size, accompanied by an increased residue-specific heterogeneity at the interface, consistent with binding-coupled changes in local mobility. Mutational analysis identifies C996 as being essential for UFD structural integrity and binding competence. Moreover, targeted alterations in the length and flexibility of the adjacent acidic loop strongly impair UbcH8 binding, demonstrating that the loop architecture is a critical determinant of efficient E2 recruitment. Together, these results provide a structural and dynamic framework for understanding E2 enzyme selection in the ISGylation pathway and highlight the role of UFD conformational dynamics in the E1-E2 complex formation.

Lysyl oxidases (LOXs) initiate the posttranslational cross-linking of collagen by oxidizing lysine to allysine residues, a process crucial for the mechanical properties of the extracellular matrix. However, dysregulated LOX levels─particularly those of the LOXL2 isoform─have been implicated in numerous fibrotic diseases and cancers. Accordingly, considerable effort has been devoted to understanding the biological role of LOXL2. Despite this interest, little is known about how the type, structure, and neighboring groups of the amine influence LOXL2 activity. Here, we determined Michaelis-Menten kinetics for a panel of lysine-based model substrates to assess the structural determinants of LOXL2-catalyzed oxidation. We show that LOXL2 oxidizes exclusively primary, α-unbranched amines. In addition, our studies revealed that an additional positively charged group enhances LOXL2 activity.

For much of the 20th century, enzyme kinetic analysis relied on deriving simplified rate equations under the steady-state approximation and later by analytical integration of differential equations for transient kinetics. This approach has since been surpassed by computational methods using numerical integration of rate equations to directly fit experimental data based on a complete user-defined model. This paradigm shift removes the constraints imposed by solving analytical equations, enabling far greater flexibility in experimental design and model complexity. Modern global fitting methods allow data from diverse experiments to be analyzed simultaneously using the minimum number of parameters supported by the information content of the data set. Global data fitting is more than just an algorithm for data analysis─it represents a fundamental change in how we design and interpret experiments, and eliminates many of the restrictions, approximations, and ambiguities inherent to equation-based analyses. In this review, we describe the principles and practice of global data fitting, compare the outcomes to conventional equation-based methods, and demonstrate its power through examples involving multiple experiments with distinct conditions and readouts. We explain why the common practice of making measurements in triplicate introduces uncertainty and we outline advanced methods for rigorously estimating errors in measurement and for establishing robust confidence limits on fitted parameters.