Molecular Pharmacology最新文献

Neuronal axons serve as a conduit for the coordinated transport of essential molecular cargo between structurally and functionally distinct subcellular compartments via axonal molecular machinery. Long-distance, efficient axonal transport of membrane-bound organelles enables signal transduction and neuronal homeostasis. Efficient axonal transport is conducted by dynein and kinesin ATPase motors that use a local ATP supply from metabolic enzymes tethered to transport vesicles. Molecular motor adaptor proteins promote the processive motility and cargo selectivity of fast axonal transport. Axonal transport impairments are directly causative or associated with many neurodegenerative diseases and neuropathologies. Cargo specificity, cargo-adaptor proteins, and posttranslational modifications of cargo, adaptor proteins, microtubules, or the motor protein subunits all contribute to the precise regulation of vesicular transit. One posttranslational lipid modification that is particularly important in neurons in regulating protein trafficking, protein-protein interactions, and protein association with lipid membranes is S-acylation. Interestingly, many fast axonal transport cargos, cytoskeletal-associated proteins, motor protein subunits, and adaptors are S-acylated to modulate axonal transport. Here, we review the established regulatory role of S-acylation in fast axonal transport and provide evidence for a broader role of S-acylation in regulating the motor-cargo complex machinery, adaptor proteins, and metabolic enzymes from low-throughput studies and S-acyl-proteomic data sets. We propose that S-acylation regulates fast axonal transport and vesicular motility through localization of the proteins required for the motile cargo-complex machinery and relate how perturbed S-acylation contributes to transport impairments in neurological disorders. SIGNIFICANCE STATEMENT: This review investigates the regulatory role of S-acylation in fast axonal transport and its connection to neurological diseases, with a focus on the emerging connections between S-acylation and the molecular motors, adaptor proteins, and metabolic enzymes that make up the trafficking machinery.

The β1-adrenergic receptor (β₁AR) is an essential G protein-coupled receptor in the heart. Its dysregulation represents a hallmark of cardiac diseases. Studies have identified a unique mode of β-arrestin interaction, where β₁AR briefly engages with β-arrestins before catalytically accumulating them at the plasma membrane (PM) independently of the receptor. Although receptor phosphorylation crucially impacts β-arrestins, the contributions of specific kinases vital in β₁AR regulation remain unclear. Here, we employed G protein-coupled receptor kinase (GRK) GRK2/3/5/6 knockout cells and the protein kinase A inhibitor H89 in bioluminescence resonance energy transfer-based assays to systematically assess GRKs and protein kinase A in direct β-arrestin2 recruitment to β₁AR and β-arrestin2 translocation to the PM. Furthermore, we compared the effects of the synthetic agonist isoprenaline with the endogenous catecholamines: epinephrine and norepinephrine. We observed pronounced differences in their kinase dependencies to mediate β-arrestin2 translocation to the PM. Upon isoprenaline stimulation, GRKs strongly influenced β-arrestin2 translocation to the PM but had no effect on direct β-arrestin2 recruitment to β₁AR. Additionally, in a GRK2-specific context, protein kinase A inhibition primarily reduced the efficacy of isoprenaline for β-arrestin2 translocation, whereas for GRK5, it decreased potency. Strikingly, these kinase-dependent effects were absent for epinephrine and norepinephrine, suggesting distinct underlying molecular mechanisms for β-arrestin2 accumulation at the PM. This observation could be explained by agonist-specific differences in receptor conformational rearrangements, as suggested by distinct changes in the NMR spectra of β₁AR. Our findings highlight that synthetic and endogenous ligands induce distinct molecular mechanisms in β₁AR regulation, emphasizing the need to consider these differences when translating molecular insights into physiological contexts. SIGNIFICANCE STATEMENT: Our findings reveal mechanistic differences in β1-adrenergic receptor-mediated catalytic activation of β-arrestin2 by synthetic and endogenous agonists, driven by distinct G protein-coupled receptor kinases and protein kinase A dependencies. Although β-arrestin2 translocation to the PM occurred to similar extents with isoprenaline, epinephrine, and norepinephrine, kinase involvement was crucial only upon Iso stimulation of β1-adrenergic receptor. By elucidating these ligand-specific pathways, this study advances our understanding of β1-adrenergic receptor signaling and regulation while additionally highlighting the importance of considering these differences when translating molecular insights into pathophysiological contexts.

Regulators of G protein signaling (RGS) proteins negatively regulate signaling through G protein-coupled receptors, and reduced RGS protein function is involved in numerous pathologies. However, therapeutic intervention is challenging, as RGS proteins lack druggable binding pockets and enzymatic activity. Instead, targeting mechanisms that control RGS protein expression show promise as an alternative. Pharmacological stabilization of RGS2 would be a feasible therapeutic strategy in pathologies associated with reduced RGS2 protein levels, such as hypertension, heart failure, and asthma. RGS2 is rapidly degraded through the ubiquitin-proteasomal system, and we recently identified the E3 ligase that recognizes RGS2. F-box Only Protein 44 (FBXO44) acts as the substrate recognition site for RGS2 in this E3 ligase complex, and we hypothesize that inhibiting the RGS2-FBXO44 interaction will lead to enhanced RGS2 levels. Here, we developed a NanoLuc Binary Technology (NanoBiT) assay that detects the interaction between RGS2 and FBXO44. This assay was used to screen 1600 compounds from the Life Chemicals protein-protein interaction fragment library. We identified a promising hit, denoted compound 10, that inhibits the RGS2-FBXO44 interaction with a potency of 19.6 μM, through direct binding to RGS2. The resulting increase in RGS2 protein levels is dependent on FBXO44, as siRNA-mediated FBXO44 knockdown attenuates the effect of compound 10. Altogether, compound 10 represents the first example of a small-molecule inhibitor of the RGS2-FBXO44 interaction and a first step toward the development of molecular probes with a defined mechanism to stabilize RGS2 protein levels. SIGNIFICANCE STATEMENT: This study provides a strategy to identify molecules that selectively inhibit RGS2 protein degradation as well as the first example of a compound with the ability to inhibit RGS2 interaction with the E3 ligase component FBXO44. This study provides proof of concept that a small-molecule RGS2-FBXO44 interaction inhibitor will increase RGS2 protein levels. Future development of compounds with this mechanism of action would be clinically useful in pathologies associated with low RGS2 protein levels, including hypertension, heart failure, and asthma.

Macrophages are major innate immune cells for the clearance of inhaled nanoparticles but may undergo cell death upon phagocytosis of certain nanoparticles due to their resistance to lysosomal degradation and high toxicity to the cell. Here we investigated the pyroptotic effect of exposure to fibrogenic multiwalled carbon nanotubes (MWCNTs) on macrophages, an inflammatory form of cell death. We first evaluated MWCNT-induced cell death in M1 and M2 macrophages that mediate the temporal inflammatory response to MWCNTs in mammalian lungs. Macrophages were differentiated from human monocytic THP-1 cells, followed by polarization to M1 or M2 cells. MWCNTs caused concentration- and time-dependent cytotoxicity in M1 and, to a lesser extent, M2 cells. Carbon black, an amorphous carbonous material control for CNTs, did not cause apparent toxicity in the cells. MWCNTs increased the production and secretion of IL-1β, accompanied by activation of caspase-1, in M1, but not M2, cells. Moreover, MWCNTs induced the formation of apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain specks and the release of cathepsin B in M1 cells, revealing activation of the nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via lysosomal damage. MWCNTs induced the cleavage of gasdermin D (GSDMD) to form the 31 kDa N-terminal fragment (GSDMD-N), the pore-forming peptide causing pyroptotic cell death. Increased IL-1β release was completely suppressed by AC-YVAD-CMK (a caspase-1 inhibitor), MCC-950 (an NLRP3 inflammasome inhibitor), or CA-074 Me (a cathepsin B inhibitor), alongside the blockage of MWCNT-induced cleavage of GSDMD. The study demonstrates that MWCNTs trigger pyroptosis in M1 macrophages and boost sterile inflammation by activating the NLRP3 inflammasome pathway. SIGNIFICANCE STATEMENT: The nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 inflammasome mediates the inflammatory response to fibrogenic nanoparticles in the lung via multiple means. The current study uncovers the induction of pyroptotic death of macrophages as a major means of nanotoxicity and sterile inflammation via the nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 pathway by nanoparticles.

Food-drug interactions occur when the presence of foods interferes with the absorption, distribution, metabolism, or excretion of pharmaceuticals. Specific compounds within foods, like certain phytochemicals from grapefruit, have been known to precipitate food-drug interactions for decades, leading to guidance from physicians and pharmacists about patients' dietary restrictions while taking certain drugs. Although approved by the Food and Drug Administration, high-intensity non-nutritive sweeteners (NNS) share qualities with drugs that suggest the potential for similar interactions. In this minireview, we have reviewed 5 of the most popular NNS, including saccharin, aspartame, acesulfame potassium, sucralose, and stevia, and detail their drug-like qualities, regulatory status, pharmacokinetics, and primary research articles containing evidence of NNS interacting with drug absorption, distribution, metabolism, and excretion. Although studies varied widely in concentration ranges for NNS, model systems, and methods, all NNS included in this review were found to have known interactions with mediators of absorption, distribution, metabolism, and excretion from studies conducted after their Food and Drug Administration approval or generally recognized as safe designation. We have highlighted essential gaps in the literature and recommend the scientific community actively research NNS as food additives that may interact with drugs. SIGNIFICANCE STATEMENT: Food-drug interactions are a growing concern in Western societies where polypharmacy and ultraprocessed foods and beverages are increasingly common. High-intensity non-nutritive sweeteners bear structural similarities to pharmaceuticals, and evidence suggests they interact with mediators of drug pharmacokinetics. This minireview highlights the interactions uncovered thus far and serves as a call to action for the scientific community to establish rigorous, consistent testing that will enable updated safety guidelines for consumers.

Dimerization and oligomerization of membrane receptors, including G protein-coupled receptors and receptor tyrosine kinases, are fundamental for regulating cell signaling and diversifying downstream responses to mediate a range of physiological processes. Receptor di/oligomers play roles in diverse facets of receptor function. Changes in receptor di/oligomers have been implicated in a range of diseases; therefore, better understanding of the specific composition and interactions between receptors in complexes is essential, especially for the development of di/oligomer-specific drugs. Previously, different optical microscopy approaches and proximity-based biophysical assays have been used to demonstrate di/oligomerization of membrane receptors. However, in recent years, single-molecule super-resolution microscopy techniques have allowed researchers to quantify and uncover the precise dynamics and stoichiometry of specific receptor complexes. This allows the organization of membrane protein receptors to be mapped across the plasma membrane to explore the effects of factors such as ligands, effectors, membrane environment, and therapeutic agents. Quantification of receptor complexes is required to better understand the intricate balance of distinct receptor complexes in cells. In this brief review, we provide an overview of single-molecule approaches for the quantification of receptor di/oligomerization. We will discuss the techniques commonly employed to study membrane receptor di/oligomerization and their relative advantages and limitations. SIGNIFICANCE STATEMENT: Receptor di/oligomerization plays an important role in their function. For some receptors, di/oligomerization is essential for functional signaling, whereas for others, it acts as a mechanism to achieve signaling pleiotropy. Aberrant receptor di/oligomerization has been implicated in a wide range of diseases. Single-molecule super-resolution microscopy techniques provide convincing methods to precisely quantify receptor complexes at the plasma membrane. Understanding receptor complex organization in disease models can also influence the targeting of specific monomeric or oligomeric complexes in therapeutic strategies.

Chemotherapy drugs such as doxorubicin (DOX) are frequently used to treat cancer, but its negative impact on the heart reduces its effectiveness. Among the members of the TRIM protein family, mitsugumin (MG)53, also known as TRIM72, is unique. It is primarily present in the plasma membrane of cardiac and skeletal muscle cells and has been demonstrated to participate in mending cellular membrane damage while protecting against heart ischemia/reperfusion injury. This research investigated the role of MG53 in DOX-induced apoptosis using H9c2 cells, a cardiomyoblast cell line, as an experimental model. Our findings indicate that DOX treatment statistically significantly upregulates MG53 expression in H9c2 cells. Furthermore, MG53 overexpression exacerbated DOX-induced apoptosis, as confirmed by elevated levels of cleaved-caspase3 and BAX and reduced expression of Bcl-2. Flow cytometry analysis supported the elevated cell death rate in cells overexpressing MG53. Additionally, MG53 overexpression was associated with reduced phosphorylation levels of protein kinase B (AKT), as indicated by the decreased phosphorylation levels of AKT. Conversely, silencing MG53 through siRNA increased the phosphorylation levels of AKT. These results imply that MG53 exacerbates DOX-induced apoptosis, related to reduced AKT phosphorylation. Our investigation sheds light on the detrimental effects of MG53 in DOX-induced myocardial damage and underscores its potential as a therapeutic target for alleviating DOX treatment-related heart toxicity. SIGNIFICANCE STATEMENT: This study reveals that mitsugumin 53 exacerbates doxorubicin-induced apoptosis in H9c2 cells, associated with altered protein kinase B signaling. Targeting mitsugumin 53 may offer a novel therapeutic approach to mitigate doxorubicin-associated cardiotoxicity.