Biochemistry Biochemistry最新文献

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that functions in pain signaling and neuroimmune communication. The CGRP receptor, CGRPR, is a class B GPCR that is a drug target for migraine headache and other disorders. Here, we used nanoBRET receptor binding and cAMP biosensor signaling assays and theoretical modeling to characterize the CGRPR "two-domain" peptide binding mechanism. Single-site extracellular domain (ECD)-binding and two-site ECD/transmembrane domain (TMD)-binding peptides were examined for CGRP and a high-affinity variant "ssCGRP" with modifications in the C-terminal region. Wildtype and ssCGRP(27-37) bound the ECD with affinities of 1 μM and 0.5 nM, and residence times of 5 s and 8 min, respectively. The (8-37) antagonist fragments had affinities of 100 nM for wildtype and 0.5 nM for ss and exhibited behavior consistent with two-site ECD/TMD binding. ssCGRP(8-37) had a residence time of 76 min. CGRP(1-37) agonist had 25-fold higher affinity for the G protein-coupled state of the CGRPR (K_i = 3 nM) than the uncoupled state (K_i = 74 nM), and elicited short-duration cAMP signaling. In contrast, ssCGRP(1-37) had similar strong affinities for both receptor states (K_i = 0.2 to 0.25 nM), and induced long-duration signaling. An equilibrium reaction network mathematical model of CGRPR activation that includes peptide and G protein binding was developed. This captured wildtype CGRP binding experiments well, but the ssCGRP binding properties were not fully reproduced, suggesting that it may exhibit a distinct binding mechanism. Together, these results advance our quantitative understanding of the CGRPR two-domain mechanism and support the ss variants as potential long-acting therapeutics.

Cell surface oligosaccharides and related polymers are commonly decorated with acyl esters that alter their structural properties and influence their interactions with other molecules. In many cases, these esters are added to polymers that are already positioned on the extracytoplasmic side of a membrane, presenting cells with a chemical challenge because the high-energy acyl donors used for these modifications are made in the cytoplasm. How activated acyl groups are passed from the cytoplasm to extra-cytoplasmic polymers has been a longstanding question. Recent mechanistic work has shown that many bacterial acyl transfer pathways operate by shuttling acyl groups through two covalent intermediates to their final destination on an extracellular polymer. Key to these and other pathways are cross-membrane acyltransferases─enzymes that catalyze transfer of acyl groups from a donor on one side of the membrane to a recipient on the other side. Here we review what has been learned recently about how cross-membrane acyltransferases in polymer acylation pathways function, highlighting the chemical and biosynthetic logic used by two key protein families, membrane-bound O-acyltransferases (MBOATs) and acyltransferase-3 (AT3) proteins. We also point out outstanding questions and avenues for further exploration.

The transcriptional regulation of metabolic genes is crucial for maintaining metabolic homeostasis under cellular stress conditions. Transcription factor 7-like 2 (TCF7l2 or TCF4) is associated with type 2 diabetes (T2D) and functions as a transcription factor for various gluconeogenic genes. T2D often coexists with metabolic dysfunction-associated steatotic liver disease (MASLD) due to common underlying mechanisms and shared risk factors such as insulin resistance and obesity. This study demonstrates the transcriptional regulation of one of the important fatty acid chain elongases implicated in T2D, HACD3 (encoded by PTPLAD1 gene), under palmitic acid (PA)-induced stress conditions. We observed that TCF7l2 is associated with histone H3K4me3-binder protein TCF19 and is corecruited to the promoter of PTPLAD1. Upon PA treatment, the TCF19-TCF7l2 complex dissociates from the lipid chain elongase gene due to the reduced level of H3K4me3 enrichment, leading to PTPLAD1 activation. Remarkably, gene expression analysis from the PA-injected mice and NAFLD patients indicates an anticorrelation whereby reduced TCF7l2 expression enhances HACD3-mediated chain elongation and triglyceride production, thereby promoting the development of MASLD. Our findings delineate that the epigenetic mechanism of activation of lipid chain elongase genes mediated by TCF7l2 in concert with TCF19 has important implications in metabolic disorders.

Phase separation is a fundamental process that enables cellular organization by forming biomolecular condensates. These assemblies regulate diverse functions by creating distinct environments, influencing reaction kinetics, and facilitating processes such as genome organization, signal transduction, and RNA metabolism. Recent studies highlight the complexity of condensate properties, shaped by intrinsic molecular features and external factors such as temperature and pH. Molecular simulations serve as an effective approach to establishing a comprehensive framework for analyzing these influences, offering high-resolution insights into condensate stability, dynamics, and material properties. This review evaluates recent advancements in biomolecular condensate simulations, with a particular focus on coarse-grained 1-bead-per-amino-acid (1BPA) protein models, and emphasizes OpenABC, a tool designed to simplify and streamline condensate simulations. OpenABC supports the implementation of various coarse-grained force fields, enabling their performance evaluation. Our benchmarking identifies inconsistencies in phase behavior predictions across force fields, even though these models accurately capture single-chain statistics. This finding underscores the need for enhanced force field accuracy, achievable through enriched training data sets, many-body potentials, and advanced optimization techniques. Such refinements could significantly improve the predictive capacity of coarse-grained models, bridging molecular details with emergent condensate behaviors.

Phase separation is a fundamental process that enables cellular organization by forming biomolecular condensates. These assemblies regulate diverse functions by creating distinct environments, influencing reaction kinetics, and facilitating processes such as genome organization, signal transduction, and RNA metabolism. Recent studies highlight the complexity of condensate properties, shaped by intrinsic molecular features and external factors such as temperature and pH. Molecular simulations serve as an effective approach to establishing a comprehensive framework for analyzing these influences, offering high-resolution insights into condensate stability, dynamics, and material properties. This review evaluates recent advancements in biomolecular condensate simulations, with a particular focus on coarse-grained 1-bead-per-amino-acid (1BPA) protein models, and emphasizes OpenABC, a tool designed to simplify and streamline condensate simulations. OpenABC supports the implementation of various coarse-grained force fields, enabling their performance evaluation. Our benchmarking identifies inconsistencies in phase behavior predictions across force fields, even though these models accurately capture single-chain statistics. This finding underscores the need for enhanced force field accuracy, achievable through enriched training data sets, many-body potentials, and advanced optimization techniques. Such refinements could significantly improve the predictive capacity of coarse-grained models, bridging molecular details with emergent condensate behaviors.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that functions in pain signaling and neuroimmune communication. The CGRP receptor, CGRPR, is a class B GPCR that is a drug target for migraine headache and other disorders. Here, we used nanoBRET receptor binding and cAMP biosensor signaling assays and theoretical modeling to characterize the CGRPR “two-domain” peptide binding mechanism. Single-site extracellular domain (ECD)-binding and two-site ECD/transmembrane domain (TMD)-binding peptides were examined for CGRP and a high-affinity variant “ssCGRP” with modifications in the C-terminal region. Wildtype and ssCGRP(27-37) bound the ECD with affinities of 1 μM and 0.5 nM, and residence times of 5 s and 8 min, respectively. The (8-37) antagonist fragments had affinities of 100 nM for wildtype and 0.5 nM for ss and exhibited behavior consistent with two-site ECD/TMD binding. ssCGRP(8-37) had a residence time of 76 min. CGRP(1-37) agonist had 25-fold higher affinity for the G protein-coupled state of the CGRPR (K_i = 3 nM) than the uncoupled state (K_i = 74 nM), and elicited short-duration cAMP signaling. In contrast, ssCGRP(1-37) had similar strong affinities for both receptor states (K_i = 0.2 to 0.25 nM), and induced long-duration signaling. An equilibrium reaction network mathematical model of CGRPR activation that includes peptide and G protein binding was developed. This captured wildtype CGRP binding experiments well, but the ssCGRP binding properties were not fully reproduced, suggesting that it may exhibit a distinct binding mechanism. Together, these results advance our quantitative understanding of the CGRPR two-domain mechanism and support the ss variants as potential long-acting therapeutics.

Time-resolved second harmonic generation (SHG) microscopy is used to investigate the physicochemical interactions between positively charged, hydrophobic, drug-like molecules and the plasma membrane of human cells (nonsmall cell lung cancer, H596). In the present study, molecular adsorption and transport of the cationic molecules, malachite green (MG) and malachite green isothiocyanate (MGITC), are studied in real time in living H596 cells and in dead, fixed H596 cells. MGITC is shown to have stronger adsorption and more rapid transport kinetics as compared to MG due to increased dipole-dipole interactions. Additionally, MGITC is found to have faster adsorption and transport kinetics in living H596 cells in comparison to fixed H596 cells, as well as higher dispersity in transport rate, pointing to changes in the nature of the plasma membrane or its integrity. Overall, the findings highlight the importance of electrostatic interactions, chemical functional groups, and cell integrity in molecular translocation dynamics across cell membranes.

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.

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective noncatechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and noncatechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, noncatechol agonists display G protein biased signaling at the D1R and have reduced or absent β-arrestin2 recruitment. A notable exception was compound (Cmpd) 19, a noncatechol agonist with full efficacy at both D1R-G protein and D1R-β-arrestin2 pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R-β-arrestin2 activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The β-arrestin2 super agonist, A-77636, showed statistically significant increases in D1R endocytosis. Moreover, β-arrestin2 recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential.

SARS-CoV-2 variant recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral targets. Mpro features several druggable sites, including active sites and allosteric sites near the dimerization interface, that regulate its catalytic activity. This study identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to the target Mpro was validated. The FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC₅₀ values in the range from 0.64 to 11.98 μM. Additionally, in vitro cell-based antiviral assays showed high efficacy with EC₅₀ values in the range of 1.51 to 18.92 μM. The crystal structure of the Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for the proteolytic activity of Mpro. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and direct the rational design of minocycline derivatives as antiviral drugs.