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1. GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade

   https://doi.org/10.1371/journal.pbio.3002685  

   Walker, Madison F; Zhang, Jingyi; Steiner, William; Ku, Pei-I; Zhu, Ju-Fen; Michaelson, Zachary; Yen, Yu-Chen; Lee, Annabel; Long, Alyssa B; Casey, Mattie J; et al (August 2024, PLOS Biology)

   Basler, Konrad (Ed.)

   During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors. 

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2. Characterization of a sperm motility signalling pathway in a gonochoric coral suggests conservation across cnidarian sexual systems

   https://doi.org/10.1098/rspb.2023.0085  

   Glass, Benjamin H.; Ashey, Jill; Okongwu, Amarachukwu R.; Putnam, Hollie M.; Barott, Katie L. (August 2023, Proceedings of the Royal Society B: Biological Sciences)

   Most stony corals liberate their gametes into the water column via broadcast spawning, where fertilization hinges upon the activation of directional sperm motility. Sperm from gonochoric and hermaphroditic corals display distinct morphological and molecular phenotypes, yet it is unknown whether the signalling pathways controlling sperm motility are also distinct between these sexual systems. Here, we addressed this knowledge gap using the gonochoric, broadcast spawning coralAstrangia poculata. We found that cytosolic alkalinization of sperm activates the pH-sensing enzyme soluble adenylyl cyclase (sAC), which is required for motility. Additionally, we demonstrate for the first time in any cnidarian that sAC activity leads to protein kinase A (PKA) activation, and that PKA activity contributes to sperm motility activation. Ultrastructures ofA. poculatasperm displayed morphological homology with other gonochoric cnidarians, and sAC exhibited broad structural and functional conservation across this phylum. These results indicate a conserved role for pH-dependent sAC-cAMP-PKA signalling in sperm motility across coral sexual systems, and suggest that the role of this pathway in sperm motility may be ancestral in metazoans. Finally, the dynamics of this pH-sensitive pathway may play a critical role in determining the sensitivity of marine invertebrate reproduction to anthropogenic ocean acidification. 

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3. Structure-Based Modeling of Environment-dependent Protonation States Across LNP Formulations with Atomistic CpHMD

   https://doi.org/10.26434/chemrxiv-2025-psmd3  

   Colston, Kyle J; Monsalve, Santiago C; Schneebeli, Severin T (March 2025, ChemRxiv)

   The pKa values and associated protonation states of ionizable lipids in lipid nanoparticle (LNP) formulations are strongly dependent on their chemical environment. This phenomenon leads to poorly understood structure-function relationships that influence payload delivery, tissue-selective biodistribution, and manufacturing. For example, the charge- and biodistribution of an mRNA-loaded LNP can vary based on the type of ionizable lipid used, the molar ratio of its components, and its cargo. Yet, the spatial resolution of experimental protonation state measurements is currently limited to the apparent charge of an ionizable lipid averaged over all environments/conformations of an LNP — best represented by its apparent pKa value. Such measurements are too coarse to capture the heterogenous charge distributions of ionizable lipids in LNPs, which influence biocorona formation and interactions with the payload. Similar limitations are inherent to classical fixed protonation-state in silico models that cannot capture the environment-dependent protonation states and pKa values determining local pKa. To address this gap in experimental and computational tools available to accurately determine the local charge distributions in LNPs, this work now incorporates a scalable continuous constant pH molecular dynamics (CpHMD) model to simulate the self-assembly processes of five reported distinct LNP formulations. Parameters for ionizable lipids were generated from fitting fixed lambda-state calculations performed with Hamiltonian replica exchange (HREX) to improve conformational sampling during parameterization. Simulated systems were composed of 100 ionizable lipids (50 mol%), cholesterol (40 mol%), distearoylphosphatidylcholine (10 mol%), and mRNA (20 nucleotides) to model the interior of an LNP. Self-assembly was simulated for 100 ns at different pH values to validate the apparent pKa for each system. The theoretically calculated apparent pKa values matched reasonably well with those measured experimentally (mean absolute error = 0.5 pKa units), and all systems exhibited pH-dependent structures. Overall, this work provides a new computational platform technology to (i) predict the pKa values of ionizable lipids in different chemical environments and (ii) enable a structure-based way to model the heterogeneous, environment-dependent charge distributions of ionizable lipids in LNP systems typically encountered during LNP manufacturing and delivery. 

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4. pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy

   https://doi.org/10.3390/molecules24030405  

   Pogostin, Brett H.; Malmendal, Anders; Londergan, Casey H.; Åkerfeldt, Karin S. (February 2019, Molecules)

   Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, 1H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine’s imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via 1H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa’s of histidine-containing peptide and protein systems. 

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5. FgSnt1 of the Set3 HDAC complex plays a key role in mediating the regulation of histone acetylation by the cAMP-PKA pathway in Fusarium graminearum

   https://doi.org/10.1371/journal.pgen.1010510  

   Gong, Chen; Xu, Daiying; Sun, Daiyuan; Kang, Jiangang; Wang, Wei; Xu, Jin-Rong; Zhang, Xue (December 2022, PLOS Genetics)

   Freitag, Michael (Ed.)

   The cAMP-PKA pathway is critical for regulating growth, differentiation, and pathogenesis in fungal pathogens. In Fusarium graminearum , mutants deleted of PKR regulatory-subunit of PKA had severe defects but often produced spontaneous suppressors. In this study eleven pkr suppressors were found to have mutations in FgSNT1 , a component of the Set3C histone deacetylase (HDAC) complex, that result in the truncation of its C-terminal region. Targeted deletion of the C-terminal 98 aa (CT98) in FgSNT1 suppressed the defects of pkr in growth and H4 acetylation. CT98 truncation also increased the interaction of FgSnt1 with Hdf1, a major HDAC in the Set3 complex. The pkr mutant had no detectable expression of the Cpk1 catalytic subunit and PKA activities, which was not suppressed by mutations in FgSNT1 . Cpk1 directly interacted with the N-terminal region of FgSnt1 and phosphorylated it at S443, a conserved PKA-phosphorylation site. CT98 of FgSnt1 carrying the S443D mutation interacted with its own N-terminal region. Expression of FgSNT1 S443D rescued the defects of pkr in growth and H4 acetylation. Therefore, phosphorylation at S443 and suppressor mutations may relieve self-inhibitory binding of FgSnt1 and increase its interaction with Hdf1 and H4 acetylation, indicating a key role of FgSnt1 in crosstalk between cAMP signaling and Set3 complex. 

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