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The Journal of Physical Chemistry... Jun 2024Desensitization of G-protein-coupled receptors (GPCR) is a general regulatory mechanism adopted by biological organisms against overstimulation of G protein signaling....
Desensitization of G-protein-coupled receptors (GPCR) is a general regulatory mechanism adopted by biological organisms against overstimulation of G protein signaling. Although the details of the mechanism are extensively studied, it is not easy to gain an overarching understanding of the process constituted by a multitude of molecular events with vastly differing time scales. To offer a semiquantitative yet predictive understanding of the mechanism, we formulate a kinetic model for the G protein signaling and desensitization by considering essential biochemical steps from ligand binding to receptor internalization. The internalization, followed by receptor depletion from the plasma membrane, attenuates the downstream signal. Together with the kinetic model and its full numerics of the expression derived for the dose-response relation, an approximated form of the expression clarifies the role played by the individual biochemical processes and allows us to identify four distinct regimes for the downregulation that emerge from the balance between phosphorylation, dephosphorylation, and the cellular level of β-arrestin.
Topics: Receptors, G-Protein-Coupled; Kinetics; Phosphorylation; Signal Transduction; beta-Arrestins; Cell Membrane; Models, Biological; Ligands
PubMed: 38832827
DOI: 10.1021/acs.jpclett.4c00967 -
ELife Jun 2024During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures....
During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
Topics: Qa-SNARE Proteins; Autophagosomes; Phosphatidylinositol Phosphates; Humans; Molecular Dynamics Simulation; Autophagy
PubMed: 38831696
DOI: 10.7554/eLife.92189 -
ACS Synthetic Biology Jun 2024Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can...
Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, -designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between "on" and "off" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.
Topics: Phosphorylation; Kinetics; Protein Processing, Post-Translational; Protein Binding; Proteins; Protein Engineering
PubMed: 38830815
DOI: 10.1021/acssynbio.4c00101 -
Molecular and Cellular Biology Jun 2024The protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) plays an important role in modulating glucose and lipid homeostasis. We...
The protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) plays an important role in modulating glucose and lipid homeostasis. We previously suggested a potential role of SHP-1 in the regulation of peroxisome proliferator-activated receptor γ2 (PPARγ2) expression and activity but the mechanisms were unexplored. PPARγ2 is the master regulator of adipogenesis, but how its activity is regulated by tyrosine phosphorylation is largely unknown. Here, we found that SHP-1 binds to PPARγ2 primarily via its N-terminal SH2-domain. We confirmed the phosphorylation of PPARγ2 on tyrosine-residue 78 (Y78), which was reduced by SHP-1 in vitro resulting in decreased PPARγ2 stability. Loss of SHP-1 led to elevated, agonist-induced expression of the classical PPARγ2 targets and , concomitant with increased lipid content in cells expressing PPARγ2, an effect blunted by abrogation of PPARγ2 phosphorylation. Collectively, we discovered that SHP-1 affects the stability of PPARγ2 through dephosphorylation thereby influencing adipogenesis.
PubMed: 38828991
DOI: 10.1080/10985549.2024.2354959 -
Frontiers in Pharmacology 2024Cardiovascular disease (CVD) is a serious public health risk, and prevention and treatment efforts are urgently needed. Effective preventive and therapeutic programs for... (Review)
Review
Cardiovascular disease (CVD) is a serious public health risk, and prevention and treatment efforts are urgently needed. Effective preventive and therapeutic programs for cardiovascular disease are still lacking, as the causes of CVD are varied and may be the result of a multifactorial combination. Mitophagy is a form of cell-selective autophagy, and there is increasing evidence that mitophagy is involved in cardioprotective processes. Recently, many studies have shown that FUN14 domain-containing protein 1 (FUNDC1) levels and phosphorylation status are highly associated with many diseases, including heart disease. Here, we review the structure and functions of FUNDC1 and the path-ways of its mediated mitophagy, and show that mitophagy can be effectively activated by dephosphorylation of Ser13 and Tyr18 sites, phosphorylation of Ser17 site and ubiquitination of Lys119 site in FUNDC1. By effectively activating or inhibiting excessive mitophagy, the quality of mitochondria can be effectively controlled. The main reason is that, on the one hand, improper clearance of mitochondria and accumulation of damaged mitochondria are avoided, and on the other hand, excessive mitophagy causing apoptosis is avoided, both serving to protect the heart. In addition, we explore the possible mechanisms by which FUNDC1-mediated mitophagy is involved in exercise preconditioning (EP) for cardioprotection. Finally, we also point out unresolved issues in FUNDC1 and its mediated mitophagy and give directions where further research may be needed.
PubMed: 38828457
DOI: 10.3389/fphar.2024.1389953 -
MedRxiv : the Preprint Server For... May 2024Aging significantly elevates the risk for Alzheimer's disease (AD), contributing to the accumulation of AD pathologies, such as amyloid-β (Aβ), inflammation, and...
Aging significantly elevates the risk for Alzheimer's disease (AD), contributing to the accumulation of AD pathologies, such as amyloid-β (Aβ), inflammation, and oxidative stress. The human prefrontal cortex (PFC) is highly vulnerable to the impacts of both aging and AD. Unveiling and understanding the molecular alterations in PFC associated with normal aging (NA) and AD is essential for elucidating the mechanisms of AD progression and developing novel therapeutics for this devastating disease. In this study, for the first time, we employed a cutting-edge spatial transcriptome platform, STOmics SpaTial Enhanced Resolution Omics-sequencing (Stereo-seq), to generate the first comprehensive, subcellular resolution spatial transcriptome atlas of the human PFC from six AD cases at various neuropathological stages and six age, sex, and ethnicity matched controls. Our analyses revealed distinct transcriptional alterations across six neocortex layers, highlighted the AD-associated disruptions in laminar architecture, and identified changes in layer-to-layer interactions as AD progresses. Further, throughout the progression from NA to various stages of AD, we discovered specific genes that were significantly upregulated in neurons experiencing high stress and in nearby non-neuronal cells, compared to cells distant from the source of stress. Notably, the cell-cell interactions between the neurons under the high stress and adjacent glial cells that promote Aβ clearance and neuroprotection were diminished in AD in response to stressors compared to NA. Through cell-type specific gene co-expression analysis, we identified three modules in excitatory and inhibitory neurons associated with neuronal protection, protein dephosphorylation, and negative regulation of Aβ plaque formation. These modules negatively correlated with AD progression, indicating a reduced capacity for toxic substance clearance in AD subject samples. Moreover, we have discovered a novel transcription factor, ZNF460, that regulates all three modules, establishing it as a potential new therapeutic target for AD. Overall, utilizing the latest spatial transcriptome platform, our study developed the first transcriptome-wide atlas with subcellular resolution for assessing the molecular alterations in the human PFC due to AD. This atlas sheds light on the potential mechanisms underlying the progression from NA to AD.
PubMed: 38826275
DOI: 10.1101/2024.05.21.24306783 -
The Journal of Biological Chemistry May 2024Adaptive immune responses comprise the activation of T cells by peptide antigens that are presented by proteins of the Major Histocompatibility Complex (MHC) on the... (Review)
Review
Adaptive immune responses comprise the activation of T cells by peptide antigens that are presented by proteins of the Major Histocompatibility Complex (MHC) on the surface of an antigen-presenting cell. As a consequence of the T cell receptor interacting productively with a certain peptide-MHC complex, a specialized cell-cell junction known as the immunological synapse forms and is accompanied by changes in the spatiotemporal patterning and function of intracellular signaling molecules. Key modifications occurring at the cytoplasmic leaflet of the plasma and internal membranes in activated T cells comprise lipid switches that affect the binding and distribution of proteins within or near the lipid bilayer. Here, we describe two major classes of lipid switches that act at this critical water/membrane interface. Phosphoinositides are derived from phosphatidylinositol, an amphiphilic molecule that contains two fatty acid chains and a phosphate group that bridges the glycerol backbone to the carbohydrate inositol. The inositol ring can be variably (de-)phosphorylated by dedicated kinases and phosphatases, thereby creating phosphoinositide signatures that define the composition and properties of signaling molecules, molecular complexes, or whole organelles. Palmitoylation refers to the reversible attachment of the fatty acid palmitate to a substrate protein's cysteine residue. DHHC enzymes, named after the four conserved amino acids in their active site, catalyze this post-translational modification and thereby change the distribution of proteins at, between, and within membranes. T cells utilize these two types of molecular switches to adjust their properties to an activation process that requires changes in motility, transport, secretion, and gene expression.
PubMed: 38823638
DOI: 10.1016/j.jbc.2024.107428 -
International Immunopharmacology Jul 2024Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca)-responsive signaling molecule,...
Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca)-responsive signaling molecule, mediates acinar cell death and inflammatory responses leading to pancreatitis. However, the detailed mechanisms for regulating CTSB activity and inflammatory cytokine production are unknown. Myricetin (MC) exhibits various biological activities, including anti-inflammatory effects. Here, we aimed to investigate MC effects on pancreatitis and the underlying mechanisms. Prophylactic and therapeutic MC treatment ameliorated the severity of cerulein-, L-arginine-, and PDL-induced acute pancreatitis (AP). The inhibition of CTSB activity by MC was mediated via decreased calcineurin activity and macrophage infiltration, not neutrophils infiltration, into the pancreas. Additionally, calcineurin activity inhibition by MC prevented the phosphorylation of Ca/CaM-dependent protein kinase kinase 2 (CaMKK2) during AP, resulting in the inhibition of CaMKIV phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) dephosphorylation. Furthermore, MC reduced nuclear factor-κB activation by modulating the calcineurin-CaMKIV-IKKα/β-Iκ-Bα and calcineurin-AMPK-sirtuin1 axes, resulting in reduced production of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Our results showed that MC alleviated AP severity by inhibiting acinar cell death and inflammatory responses, suggesting that MC as a calcineurin and CaMKK2 signaling modulator may be a potential treatment for AP.
Topics: Animals; Pancreatitis; Flavonoids; Cytokines; Cathepsin B; Mice; Male; Mice, Inbred C57BL; Calcineurin; Anti-Inflammatory Agents; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Ceruletide; NF-kappa B; Pancreas; Signal Transduction; Arginine; Disease Models, Animal; AMP-Activated Protein Kinases
PubMed: 38823179
DOI: 10.1016/j.intimp.2024.112284 -
Acta Neuropathologica Communications May 2024Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies....
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
Topics: Animals; Humans; Male; Mice; Alkaline Phosphatase; alpha-Synuclein; Brain; Mice, Inbred C57BL; Mice, Transgenic; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Synucleinopathies
PubMed: 38822421
DOI: 10.1186/s40478-024-01785-0 -
Nature Communications May 2024The two-pore domain potassium (K) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and...
The two-pore domain potassium (K) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and phosphorylation. This regulation involves the C-terminus as a polymodal stimulus sensor and the selectivity filter (SF) as channel gate. Using crystallographic up- and down-state structures of TREK-2 as a template for full atomistic molecular dynamics (MD) simulations, we reveal that the SF in down-state undergoes inactivation via conformational changes, while the up-state structure maintains a stable and conductive SF. This suggests an atomistic mechanism for the low channel activity previously assigned to the down state, but not evident from the crystal structure. Furthermore, experimentally by using (de-)phosphorylation mimics and chemically attaching lipid tethers to the proximal C-terminus (pCt), we confirm the hypothesis that moving the pCt towards the membrane induces the up-state. Based on MD simulations, we propose two gating pathways by which movement of the pCt controls the stability (i.e., conductivity) of the filter gate. Together, these findings provide atomistic insights into the SF gating mechanism and the physiological regulation of TREK channels by phosphorylation.
Topics: Potassium Channels, Tandem Pore Domain; Molecular Dynamics Simulation; Humans; Phosphorylation; Ion Channel Gating; Protein Domains; Cytosol; Animals; HEK293 Cells; Crystallography, X-Ray
PubMed: 38821927
DOI: 10.1038/s41467-024-48823-y