Biochemistry Biochemistry最新文献

Molecular-level structural polymorphisms of β-amyloid (Aβ) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aβ peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aβ concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aβ (Aβ₄₀) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aβ₄₀.

(2S,3S)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH₃ group is substituted by a CH₂F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a ¹³C-labeled CH₂F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH₂F groups invariably populate staggered rotamers. Most CH₂F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. ¹⁹F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted ¹⁹F resonances correlate with large ³J_HF coupling constants, confirming the impact of the γ-gauche effect on the signal dispersion. The present work is the second experimental verification of the effect and extends its validity to fluorovaline. The abundance of valine in proteins and structural conservation with FVal renders this valine analogue attractive for probing proteins by ¹⁹F NMR spectroscopy.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (k_cat/K_M = 1.63 × 10³ M^-1 s^-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (k_I = 0.00325 s^-1; t_1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Utilizing induced pluripotent stem cells (iPSCs) in drug screening and cell replacement therapy has emerged as a method with revolutionary applications. With the advent of patient-specific iPSCs and the subsequent development of cells that exhibit disease phenotypes, the focus of medication research will now shift toward the pathology of human diseases. Regular iPSCs can also be utilized to generate cells that assess the negative impacts of medications. These cells provide a much more precise and cost-efficient approach compared to many animal models. In this review, we explore the utilization of small-molecule drugs to enhance the growth of iPSCs and gain insights into the process of reprogramming. We mainly focus on the functions of small molecules in modulating different signaling pathways, thereby modulating cell fate. Understanding the way small molecule drugs interact with iPSC technology has the potential to significantly enhance the understanding of physiological pathways in stem cells and practical applications of iPSC-based therapy and screening systems, revolutionizing the treatment of diseases.

In a recent study published in Nature, Malamud et al. identified how neutrophil MICL recognizes neutrophil extracellular traps (NETs). This recognition suppresses further neutrophil activation and NET production, thereby preventing a vicious cycle of inflammation.

While a balanced intake of macronutrients — carbohydrates, fats, and proteins — is essential for metabolic homeostasis, animals need higher protein intake during critical life stages like pregnancy. A recent paper in Cell by Wu et al. introduces the novel concept of adjusting protein intake setpoints based on sex and mating status, using two opposing G protein-coupled receptor (GPCR) signaling pathways that regulate protein appetite-controlling neurons in the fruit fly, Drosophila melanogaster.

The kinetics of the interaction between Musashi-1 (MSI1) and RNA have been characterized using surface plasmon resonance biosensor analysis. Truncated variants of human MSI1 encompassing the two homologous RNA recognition motifs (RRM1 and RRM2) in tandem (aa 1–200), and the two RRMs in isolation (aa 1–103 and aa 104–200, respectively) were produced. The proteins were injected over sensor surfaces with immobilized RNA, varying in sequence and length, and with one or two RRM binding motifs. The interactions of the individual RRMs with all RNA variants were well described by a 1:1 interaction model. The interaction between the MSI1 variant encompassing both RRM motifs was bivalent and rapid for all RNA variants. Due to difficulties in fitting this complex data using standard procedures, we devised a new method to quantify the interactions. It revealed that two RRMs in tandem resulted in a significantly longer residence time than a single RRM. It also showed that RNA with double UAG binding motifs and potential hairpin structures forms less stable bivalent complexes with MSI1 than the single UAG motif containing linear RNA. Substituting the UAG binding motif with a CAG sequence resulted in a reduction of the affinity of the individual RRMs, but for MSI1, this reduction was strongly enhanced, demonstrating the importance of bivalency for specificity. This study has provided new insights into the interaction between MSI1 and RNA and an understanding of how individual domains contribute to the overall interaction. It provides an explanation for why many RNA-binding proteins contain dual RRMs.