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Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson's disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the "CC" motif. Among all human protein kinases, only LRRK2 contains this "CC" motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S-) in GaMD simulations allowed us to analyse how each of the cysteines in the "CC" motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.

Abstract Severe febrile illnesses in children encompass life-threatening organ dysfunction caused by diverse pathogens and other severe inflammatory syndromes. A comparative approach to these illnesses may identify shared and distinct features of host immune dysfunction amenable to immunomodulation. Here, using immunophenotyping with mass cytometry and cell stimulation experiments, we illustrate trajectories of immune dysfunction in 74 children with multi-system inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, 30 with bacterial infection, 16 with viral infection, 8 with Kawasaki disease, and 42 controls. We explore these findings in a secondary cohort of 500 children with these illnesses and 134 controls. We show that neutrophil activation and apoptosis are prominent in multi-system inflammatory syndrome, and that this is partially shared with bacterial infection. We show that memory T cells from patients with multi-system inflammatory syndrome and bacterial infection are exhausted. In contrast, we show viral infection to be characterized by a distinct signature of decreased interferon signaling and lower interferon receptor gene expression. Improved understanding of immune dysfunction may improve approaches to immunomodulator therapy in severe febrile illnesses in children.

Abstract Background Whole blood host transcript signatures show great potential for diagnosis of infectious and inflammatory illness, with most published signatures performing binary classification tasks. Barriers to clinical implementation include validation studies, and development of strategies that enable simultaneous, multiclass diagnosis of febrile illness based on gene expression. Methods We validated five distinct diagnostic signatures for paediatric infectious diseases in parallel using a single NanoString nCounter® experiment. We included a novel 3-transcript signature for childhood tuberculosis, and four published signatures which differentiate bacterial infection, viral infection, or Kawasaki disease from other febrile illnesses. Signature performance was assessed using receiver operating characteristic curve statistics. We also explored conceptual frameworks for multiclass diagnostic signatures, including additional transcripts found to be significantly differentially expressed in previous studies. Relaxed, regularised logistic regression models were used to derive two novel multiclass signatures: a mixed One-vs-All model (MOVA), running multiple binomial models in parallel, and a full-multiclass model. In-sample performance of these models was compared using radar-plots and confusion matrix statistics. Results Samples from 91 children were included in the study: 23 bacterial infections (DB), 20 viral infections (DV), 14 Kawasaki disease (KD), 18 tuberculosis disease (TB), and 16 healthy controls. The five signatures tested demonstrated cross-platform performance similar to their primary discovery-validation cohorts. The signatures could differentiate: KD from other diseases with area under ROC curve (AUC) of 0.897 [95% confidence interval: 0.822–0.972]; DB from DV with AUC of 0.825 [0.691–0.959] (signature-1) and 0.867 [0.753–0.982] (signature-2); TB from other diseases with AUC of 0.882 [0.787–0.977] (novel signature); TB from healthy children with AUC of 0.910 [0.808–1.000]. Application of signatures outside of their designed context reduced performance. In-sample error rates for the multiclass models were 13.3% for the MOVA model and 0.0% for the full-multiclass model. The MOVA model misclassified DB cases most frequently (18.7%) and TB cases least (2.7%). Conclusions Our study demonstrates the feasibility of NanoString technology for cross-platform validation of multiple transcriptomic signatures in parallel. This external cohort validated performance of all five signatures, including a novel sparse TB signature. Two exploratory multi-class models showed high potential accuracy across four distinct diagnostic groups.

Abstract The effects of subthalamic nucleus deep brain stimulation (STN-DBS) on anxiety in Parkinson’s disease (PD) are understudied. We identified clinical predictors of STN-DBS effects on anxiety in this study. In this prospective, open-label, multicentre study, we assessed patients with anxiety undergoing STN-DBS for PD preoperatively and at 6-month follow-up postoperatively. We assessed the Hospital Anxiety and Depression Scale (HADS-anxiety and depression subscales), Unified PD Rating Scale-motor examination, Scales for Outcomes in PD-motor (SCOPA-M)-activities of daily living (ADL) and -motor complications, Non-Motor Symptom Scale (NMSS), PDQuestionnaire-8 (PDQ-8), and levodopa-equivalent daily dose. We tested changes at follow-up with Wilcoxon signed-rank test and corrected for multiple comparisons (Bonferroni method). We identified patients with a clinically relevant anxiety improvement of anxiety based on a designated threshold of ½ standard deviation of baseline HADS-anxiety. Moreover, we investigated predictors of HADS-anxiety changes with correlations and linear regressions. We included 50 patients with clinically relevant baseline anxiety (i.e., HADS-anxiety ≥ 8) aged 63.1 years ± 8.3 with 10.4 years ± 4.5 PD duration. HADS-anxiety improved significantly at 6-month follow-up as 80% of our cohort experienced clinically relevant anxiety improvement. In predictor analyses, worse baseline SCOPA-ADL and NMSS-urinary domain were associated with greater HADS-anxiety improvements. HADS-anxiety and PDQ-8 changes correlated moderately. Worse preoperative ADL and urinary symptoms predicted favourable postoperative anxiety outcome, which in turn was directly proportionate to greater QoL improvement. This study highlights the importance of detailed anxiety assessments alongside other non-motor and motor symptoms when advising and monitoring patients undergoing STN-DBS for PD.

BackgroundTo identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections.MethodsChildren presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39).ResultsIn the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV.ConclusionsMIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW   and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.

Background Patients with Kawasaki disease (KD) with coronary artery involvement require long‐term cardiac care. Although respective evidence‐based recommendations are missing, cardiac catheterization is still considered the gold standard for diagnosing detailed coronary pathology. Therefore, to better understand coronary artery pathology development, we conducted a survey to document and evaluate cardiac catheterization data in a European population. Methods and Results We retrospectively analyzed cardiac catheterization data from KD children from the year 2010 until April 2023. This registry covers basic acute‐phase clinical data, and more importantly, detailed information on morphology, distribution, and the development of coronary artery pathologies. A total of 164 mainly White patients (65% boys) were included. A relevant number of patients had no coronary artery aneurysm (CAA) at the cardiac catheterization, indicating that distal CAAs were almost exclusively detected alongside proximal CAAs. Patients with multiple CAAs revealed a significant positive correlation between the number of CAAs and their dimensions in diameter and in length. Location of the CAA within the coronary artery, age at onset of KD, or natal sex did not significantly influence CAA diameters, but CAAs were longer in older children and in boys. Conclusions That distal CAAs were only present together with proximal ones will hopefully reduce diagnostic CCs in patients with KD without echocardiographically detected proximal CAAs. Furthermore, this study gives valuable insights into dimensional specifics of CAAs in patients with KD. As an ongoing registry, future analyses will further explore long‐term outcomes and performed treatments, helping to refine clinical long‐term strategies for patients with KD. REGISTRATION URL: https://drks.de/; Unique Identifier: DRKS00031022.

Although Fischers extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

BackgroundMultisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments.MethodsThe Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370.FindingsWe enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2-11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75-1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58-1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91-1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70-1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11-0·20]; p

Abstract Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson’s disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the “CC” motif. Among all human protein kinases, only LRRK2 contains this “CC” motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S-) in GaMD simulations allowed us to analyse how each of the cysteines in the “CC” motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.

Abstract Background A frequent complication of Fontan operations is unilateral diaphragmatic paresis, which leads to hemodynamic deterioration of the Fontan circulation. A potential new therapeutic option is the unilateral diaphragmatic pacemaker. In this study, we investigated the most effective stimulation location for a potential fully implantable system in a porcine model. Methods Five pigs (20.8 ± 0.95 kg) underwent implantation of a customized cuff electrode placed around the right phrenic nerve. A bipolar myocardial pacing electrode was sutured adjacent to the motor point and peripherally at the costophrenic angle (peripheral diaphragmatic muscle). The electrodes were stimulated 30 times per minute with a pulse duration of 200 µs and a stimulation time of 300 ms. Current intensity was the only variable changed during the experiment. Results Effective stimulation occurred at 0.26 ± 0.024 mA at the phrenic nerve and 7 ± 1.22 mA at the motor point, a significant difference in amperage (p = 0.005). Even with a maximum stimulation of 10 mA at the peripheral diaphragm muscle, however, no effective stimulation was observed. Conclusion The phrenic nerve seems to be the best location for direct stimulation by a unilateral thoracic diaphragm pacemaker in terms of the required amperage level in a porcine model.

BackgroundKawasaki disease (KD) is a systemic vasculitis that mainly affects children under 5 years of age. Up to 30% of patients develop coronary artery abnormalities, which are reduced with early treatment. Timely diagnosis of KD is challenging but may become more straightforward with the recent discovery of a whole-blood host response classifier that discriminates KD patients from patients with other febrile conditions. Here, we bridged this microarray-based classifier to a clinically applicable quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay: the Kawasaki Disease Gene Expression Profiling (KiDs-GEP) classifier.MethodsWe designed and optimized a qRT-PCR assay and applied it to a subset of samples previously used for the classifier discovery to reweight the original classifier.ResultsThe performance of the KiDs-GEP classifier was comparable to the original classifier with a cross-validated area under the ROC curve of 0.964 [95% CI: 0.924-1.00] vs 0.992 [95% CI: 0.978-1.00], respectively. Both classifiers demonstrated similar trends over various disease conditions, with the clearest distinction between individuals diagnosed with KD vs viral infections.ConclusionWe successfully bridged the microarray-based classifier into the KiDs-GEP classifier, a more rapid and more cost-efficient qRT-PCR assay, bringing a diagnostic test for KD closer to the hospital clinical laboratory.ImpactA diagnostic test is needed for Kawasaki disease and is currently not available. We describe the development of a One-Step multiplex qRT-PCR assay and the subsequent modification (i.e., bridging) of the microarray-based host response classifier previously described by Wright et al. The bridged KiDs-GEP classifier performs well in discriminating Kawasaki disease patients from febrile controls. This host response clinical test for Kawasaki disease can be adapted to the hospital clinical laboratory.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilised 1H-NMR spectroscopy-based metabolomics, in situ enzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: (1) the mini-Krebs-cycle, fuelled by glutamine and branched chain amino acids, generating N-acetylaspartate; (2) the alanine-generating Cahill-cycle; (3) the lactate-releasing Cori-cycle. Moreover, the metabolomics data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.

This review highlights the importance of extracellular matrix (ECM) biomaterials in understanding the biology of human trabecular meshwork (TM) and Schlemm's canal (SC) cells under normal and simulated glaucoma-like conditions. We provide an overview of recent progress in the development and application of state-of-the-art 3D ECM biomaterials including cell-derived ECM, ECM scaffolds, Matrigel, and ECM hydrogels for studies of TM and SC cell (patho)biology. Such bioengineered platforms enable accurate and reliable modeling of tissue-like cell-cell and cell-ECM interactions. They bridge the gap between conventional 2D approaches and in vivo/ex vivo models, and have the potential to aid in the identification of the causal mechanism(s) for outflow dysfunction in ocular hypertensive glaucoma. We discuss each model's benefits and limitations, and close with an outlook on future directions.

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, Cα and Cβ, and four functionally nonredundant R-subunits (RIα, RIβ, RIIα, RIIβ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, Cβ actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the Cα1-subunit, we know almost nothing about these Cβ isoforms wherein Cβ2 is highly expressed in lymphocytes, and Cβ3 and Cβ4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the Cβ proteins appear to be functionally important and nonredundant with the Cα isoforms. Imaging in retina also supports nonredundant roles for Cβ as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the Cα- and Cβ-subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

Kawasaki disease (KD) is a paediatric vasculitis associated with coronary artery aneurysms (CAA). Genetic variants influencing susceptibility to KD have been previously identified, but no risk alleles have been validated that influence CAA formation. We conducted a genome-wide association study (GWAS) for CAA in KD patients of European descent with 200 cases and 276 controls. A second GWAS for susceptibility pooled KD cases with healthy paediatric controls from vaccine trials in the UK (n = 1609). Logistic regression mixed models were used for both GWASs. The susceptibility GWAS was meta-analysed with 400 KD cases and 6101 controls from a previous European GWAS, these results were further meta-analysed with Japanese GWASs at two putative loci. The CAA GWAS identified an intergenic region of chromosome 20q13 with multiple SNVs showing genome-wide significance. The risk allele of the most associated SNV (rs6017006) was present in 13% of cases and 4% of controls; in East Asian 1000 Genomes data, the allele was absent or rare. Susceptibility GWAS with meta-analysis with previously published European data identified two previously associated loci (ITPKC and FCGR2A). Further meta-analysis with Japanese GWAS summary data from the CASP3 and FAM167A genomic regions validated these loci in Europeans showing consistent effects of the top SNVs in both populations. We identified a novel locus for CAA in KD patients of European descent. The results suggest that different genes determine susceptibility to KD and development of CAA and future work should focus on the function of the intergenic region on chromosome 20q13.