Biochemistry Biochemistry最新文献

Aggregation of the tau protein into cross-β amyloid fibrils is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Developing small molecules that bind these tau fibrils is important for the diagnosis and treatment of tauopathies. Here, we report the binding sites of a positron emission tomography (PET) ligand, PI-2620, to a recombinant tau construct that adopts the C-shaped AD fold. Using solid-state NMR ¹³C-¹⁹F rotational-echo double-resonance (REDOR) experiments, we measured the proximity of protein residues to the fluorine atom of the ligand. These data indicate that PI-2620 binds at two main locations in the concave interior of the C-shaped structure. Molecular docking simulations constrained by these REDOR data identified five binding poses at these two locations. In addition, 2D ¹³C-¹³C correlation NMR spectra indicate that PI-2620 decreased the intensities of residues at the protofilament interfaces, indicating that the ligand disordered the filament packing. Quantitative analysis of the ¹⁹F NMR spectra indicates that PI-2620 binds these AD-fold tau fibrils with a stoichiometry of ∼20 mol %, in which 10 mol % are immobilized and the rest are mobile. These results provide experimental constraints to the interaction of this second-generation PET tracer with tau fibrils adopting the AD fold and should be useful for the development of future imaging agents with improved stoichiometry and specificity for AD tau.

Biased signaling has transformed pharmacology by revealing that receptors, particularly G protein-coupled receptors (GPCRs), can activate specific intracellular pathways selectively rather than uniformly. This discovery enables the development of targeted therapeutics that minimize side effects by precisely modulating receptor activity. Functionally selective ligands, which preferentially activate distinct signaling branches, have become essential tools for exploring receptor mechanisms and uncovering the complexities of GPCR signaling. These ligands help clarify receptor function in various physiological and pathological contexts, offering profound implications for therapeutic innovation. GPCRs, which mediate a wide range of cellular responses through coupling to G proteins and arrestins, are key pharmacological targets, with nearly a third of FDA-approved drugs acting on them. Recent advancements in biosensor development, multiplex assay platforms, and deep mutational scanning methods are improving our ability to define GPCR signaling, allowing for a better understanding of biased signaling pathways.

Organismal aging is accompanied by the accumulation of senescent cells in the body, which drives tissue dysfunction. Senescent cells have a distinctive profile, including proliferation arrest, resistance to apoptosis, altered gene expression, and high inflammation. Despite global signaling and metabolic dysregulation during senescence, the underlying reasons for changes in signaling remain unclear. GPCRs are pivotal in cellular signaling, dynamically mediating the complex interplay between cells and their surrounding environment to maintain cellular homeostasis. The chemokine receptor CXCR4 plays a crucial role in modulating immune responses and inflammation. It has been shown that the expression of CXCR4 increases in cells undergoing senescence, which enhances inflammation postactivation. Here, we examine CXCR4 signaling in deeply senescent cells (aged cells), where cholesterol and its oxidized derivatives, oxysterols, affect receptor function. We report elevated oxysterol levels in senescent cells, which altered classical CXCL12-mediated CXCR4 signaling. Tail-oxidized sterols disrupted signaling more than ring-oxidized counterparts. Molecular dynamics simulations revealed that 27-hydroxycholesterol displaces cholesterol and binds strongly to alter the conformation of critical signaling residues, modifying the sterol-CXCR4 interaction landscape. Our study provides a molecular view of the observed mitigated GPCR signaling in the presence of oxysterols, which switched G-protein signaling from Gα_i/o to Gα_s class. Overall, we present an altered paradigm of GPCR signaling, where cholesterol oxidation alters the signaling outcome in aged cells.

Glycosylated macrolactones (macrolides) often display broad and potent biological activities and are targets for drug development and discovery. The modular genetic organization of macrolide polyketide synthases (PKSs) and various polyketide tailoring enzymes has inspired the combinatorial biosynthesis of new-to-nature macrolides. However, most engineered PKS and macrolide biosynthetic pathways are ineffective and produce reduced or negligible product titers. Directed evolution could improve the activity of engineered PKSs and associated pathways but critically requires a high-throughput screen to identify active variants from large libraries. Transcription factor-based biosensors can be used for this purpose. However, the effector specificity of the only known macrolide-sensing transcription factor MphR is limited to macrolides modified with the sugar, desosamine. The potential applications of MphR are subsequently limited, ruling out the possibility of leveraging MphR to screen libraries of pathway variants that make macrolactones that lack sugars (i.e., macrolide aglycones) such as the direct products of PKSs. In this study, we aimed to engineer the effector specificity of the MphR biosensor strain for detecting macrolide aglycones. By developing an "effector walking" strategy coupled with efflux pump deletion, the effector profile of MphR was dramatically broadened to include several erythronolide macrolactones. This work sets the stage for applying directed evolution and other high-throughput screening approaches to various PKSs. Our results suggest a broadly applicable approach to developing biosensors that detect ligands that are very different in structure from the native effector.

Base editing is a common mechanism by which organisms expand their genetic repertoire to access new functions. Here, we explore the mechanism of tRNA recognition in the bacterial deaminase TadA, which exclusively recognizes tRNA^Arg2 and converts the wobble base adenosine (A34) to inosine. We quantitatively evaluate the dynamics of tRNA binding by incorporating the fluorescent adenine analogue 2-aminopurine (2-AP) at position 34 in the wobble base of the anticodon loop. Time-resolved fluorescence and anisotropy studies revealed that the recognition process is finely tuned. Mutations in residues directly involved in facilitating deamination, such as E55A and N42A, showed a minimal impact on binding dynamics. In contrast, mutations in the "capping residues", notably R149, unique to prokaryotic TadAs and located 12-15 Å away from the catalytic center, significantly disrupted binding and consequently catalytic activity. The capping residues play a critical role in enabling tRNA recognition, thereby underscoring their importance in enzyme function. Moreover, for effective catalysis, peripheral positively charged residues (R70, R94) that are part of the adjacent subunit in the dimeric assembly are important to splay out the tRNA, assisting in A34 attaining a flipped-out conformation. Perturbations in these extended regions, although 15-20 Å away from the active site, disrupt the binding dynamics and consequently the function, emphasizing the fine regulation of the tRNA recognition process. Analysis reveals that the C-terminal end of the extended helix where R149 is positioned, acts as a selectivity filter imparting exclusivity toward the deamination of tRNA^Arg2 by TadA.

Antimicrobial peptides (AMPs) have emerged as a promising solution to the escalating public health threat caused by multidrug-resistant bacteria. Although ongoing research efforts have established AMP's role in membrane permeabilization and leakage, the precise mechanisms driving these disruption patterns remain unclear. We leverage molecular dynamics (MD) simulations enhanced by membrane mimetic (HMMM) to systematically investigate how the physiochemical properties of magainin (+3) and pexiganan (+9) affect their localization, insertion, curvature perturbation, and membrane binding ensemble. Building on existing microbiology, NMR, circular dichroism, and fluorescence data, our analysis reveals that the lipid makeup is a key determinant in the binding dynamics and structural conformation of AMPs. We find that phospholipid type is crucial for peptide localization, demonstrated through magainin's predominant interaction with lipid tails and pexiganan's with polar headgroups in POPC/POPS membranes. The membrane curvature changes induced by pexiganan relative to magainin suggest that AMPs with larger charges have more potential in modulating bilayer bending. These insights advance our understanding of AMP-membrane interactions at the molecular level, offering guidance for the design of targeted antimicrobial therapies.

Intrinsically disordered proteins (IDPs) are key components of cellular signaling and regulatory networks. They frequently remain dynamic even in complexes and thus rely on potentially subtle shifts in the disordered conformational ensemble for function. Understanding the molecular basis of these fascinating mechanisms of IDP function and regulation requires a detailed characterization of dynamic ensembles in various biologically relevant states. Here, we study the phosphorylation dependence of the dynamic interaction between the N-terminal transactivation domain (NTAD) and DNA-binding domain (DBD) of tumor suppressor p53, which plays a key role in the autoinhibition and regulation of p53 activation or termination during various stages of stress response. By extending the hybrid-resolution (HyRes) coarse-grained (CG) protein force field to model phosphorylated side chains, we show that HyRes simulations accurately recapitulate the effects of phosphorylation on the p53 NTAD/DBD interactions. The simulated ensembles show that phosphorylation of Thr55 as well as Ser46 enhances dynamic NTAD/DBD interactions and further induces conformational shifts that promote trans interactions between two p53 dimers to drive dissociation from DNA. These CG simulations thus provide a strong molecular basis in support of previous experimental studies suggesting the central role of dynamic interactions of disordered domains and phosphorylation in the function of p53. The success of this study also suggests that HyRes provides an efficient and viable tool for studying dynamic interactions and post-translational modifications in IDP function and regulation.

Gp2.5, an essential single-stranded DNA-binding protein encoded by bacteriophage T7, is integral to various steps of DNA metabolism. Unlike other single-stranded DNA binding proteins, it greatly facilitates the annealing of complementary DNA strands. Gp2.5 efficiently anneals DNA duplexes as short as 30 base pairs: efficient annealing occurs at a 100-fold lower concentration of complementary strands than that required in the absence of gp2.5. Additionally, gp2.5 selectively promotes DNA annealing with no observed effect on RNA or DNA hybrids. Kinetic studies show a substantial increase in the annealing rate, with gp2.5 accelerating the process by 30-fold compared with spontaneous annealing. Gp2.5 tolerates mismatches and unpaired loops within DNA, facilitating annealing in sequences with slight imperfections. FRET analysis demonstrates that gp2.5 brings strands of ssDNA into close proximity irrespective of their complementarity, likely through interactions between gp2.5 molecules. A unique long α helix A in gp2.5 is critical for its annealing activity: deletions of helix A impair DNA annealing without affecting DNA replication functions.

In the innate immune system, the CD33 receptor modulates microglial activity. Its downregulation promises to slow Alzheimer's disease, and it is already targeted in blood cancers. The mechanism underlying CD33 signaling is unresolved. Starting from the available crystal structure of its extracellular IgV-IgC1 domains, we have assembled a model of the human CD33 receptor by characterizing the oligomerization and structure of IgC1, transmembrane, and cytosolic domains in solution. IgC1 homodimerizes via intermolecular β-strand pairing and packing. In contrast, the 21-residue transmembrane helix of CD33 appears monomeric and straight, with a conserved thin neck and thick belly appearance followed by a positively charged cytosolic patch. The cytosolic domain is dynamically unstructured. Sequence alignment and AlphaFold models indicate that IgC domains in the family of human Siglecs, to which CD33 belongs, are surprisingly variable. Only Siglec-6 is identified to analogously dimerize via IgC1. Our CD33 structural model suggests that the receptor is not signaling via a monomer-dimer shift. Rather, we propose that, aided but also constrained by dimerization, multivalent ligands may concentrate the receptor transmembrane and cytosolic domains sufficiently to trigger colocalization with an activating kinase.

Rare diseases are a diverse group of disorders that, despite each individual condition’s rarity, collectively affect a significant portion of the global population. Currently approximately 10,000 rare diseases exist globally, with 80% of these diseases being identified as having genetic origins. In this Review, we examine data from the CAS Content Collection to summarize scientific progress in the area of rare diseases. We examine the publication landscape in the area in an effort to provide insights into current advances and developments. We then discuss the evolution of key concepts in the field, genetic associations, as well as the major technologies and development pipelines of rare disease treatments. We focus our attention on three specific rare diseases: (i) amyotrophic lateral sclerosis, a terminal neurodegenerative disease affecting the central nervous system resulting in progressive loss of motor neurons that control voluntary muscles; (ii) Huntington’s disease, another terminal neurodegenerative disease that causes progressive degeneration of nerve cells in the brain, with a wide impact on a person’s functional abilities; and (iii) myasthenia gravis, a chronic autoimmune synaptopathy leading to skeletal muscle weakness. While the pathogenesis of these rare diseases is being elucidated, there is neither a cure nor preventative treatment available, only symptomatic treatment. The objective of the paper is to provide a broad overview of the evolving landscape of current knowledge on rare diseases and specifically on the biology and genetics of the three spotlighted diseases, to outline challenges and evaluate growth opportunities, an aim to further efforts in solving the remaining challenges.