RedOx regulation of LRRK2 kinase activity by active site cysteines.

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.

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

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.

Gauging Dynamics-driven Allostery Using a New Computational Tool: A CAP Case Study.

In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAP's entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids.

Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs' unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase's (PylRS') remarkable substrate polyspecificity, this work attempts to increase PylRS' ability in directly charging tRNAPyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins' physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCß-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Bladder Distention Demonstrating Vesicourachal Diverticulum on 18 F-FDG PET/CT Images of a Lung Cancer Patient.

ABSTRACT: A 58-year-old man with lung cancer was referred for an 18 F-FDG PET/CT scan for pretreatment staging. The FDG PET/CT scan revealed focal uptakes in the lower abdomen. We differentiated the etiology of the lesions by performing a delayed scan with urine retention and bladder distension. The delayed scan demonstrated a tubular, radioactivity-filled structure arising above the urinary bladder. Combining the FDG PET/CT scan, clinical findings, and ultrasonography, we made the diagnosis of vesicourachal diverticulum.

Capturing the domain crosstalk in full length LRRK2 and LRRK2RCKW.

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.

Capturing Differences in the Regulation of LRRK2 Dynamics and Conformational States by Small Molecule Kinase Inhibitors.

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson's disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2RCKW, leading to a more compact LRRK2RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (PKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent accumulation of aberrantly active enzyme. Here, we examine how a single residue in the C1A domain of PKCß, arginine 42 (R42), permits quality-control degradation when mutated to histidine in cancer (R42H) and blocks downregulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (R42P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and downregulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity to that of WT. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Novel LRR-ROC Motif That Links the N- and C-terminal Domains in LRRK2 Undergoes an Order-Disorder Transition Upon Activation.

Mutations in LRRK2, a large multi-domain protein kinase, create risk factors for Parkinson's Disease (PD). LRRK2 has seven well-folded domains that include three N-terminal scaffold domains (NtDs) and four C-terminal domains (CtDs). In full-length inactive LRRK2 there is an additional well-folded motif, the LRR-ROC Linker, that lies between the NtDs and the CtDs. This motif, which is stabilized by hydrophobic residues in the LRR and ROC/COR-A domains, is anchored to the C-Lobe of the kinase domain. The LRR-ROC Linker becomes disordered when the NtDs are unleashed from the CtDs following activation by Rab29 or by various PD mutations. A key residue within the LRR-ROC Linker, W1295, sterically blocks access of substrate proteins. The W1295A mutant blocks cis-autophosphorylation of S1292 and reduces phosphorylation of heterologous Rab substrates. GaMD simulations show that the LRR-Linker motif, P + 1 loop and the inhibitory helix in the DYGψ motif are very stable. Finally, in full-length inactive LRRK2 ATP is bound to the kinase domain and GDP:Mg to the GTPase/ROC domain. The fundamentally different mechanisms for binding nucleotide (G-Loop vs P-Loop) are captured by these GaMD simulations. In this model, where ATP binds with low affinity (µM range) to N-Lobe capping residues, the known auto-phosphorylation sites are located in the space that is sampled by the flexible phosphates thus providing a potential mechanism for cis-autophosphorylation.

18F-FDG PET/CT Did Not Increase the Risk of Cataract Occurrence in Oncology Patients: A Nationwide Population-Based Cohort Study.

This study aimed to evaluate the risk of cataract formation associated with radiation exposure from 18F-FDG PET/CT in oncology patients, using data from Taiwan's National Health Insurance Research Database. The exposed group (Group E) consisted of oncology patients receiving 18F-FDG PET/CT within the first year of a cancer diagnosis. The comparison group (Group C) included subjects who had never been exposed to 18F-FDG PET/CT radiation and were propensity score-matched by date of enrolment, age, sex, cancer type, associated comorbidities, and CT utilization. Multiple Cox proportional hazard regression analysis was used to estimate the hazard ratio (HR) of cataract risk due to radiation exposure, while adjusting for potential confounding factors. A total of 703 patients and 1406 matched subjects were in Groups E and C, respectively. The incidence of cataract formation was not significantly higher among subjects in Group E (adjusted HR = 1.264; 95% confidence interval [CI] = 0.845-1.891). Our results revealed that 18F-FDG PET/CT was not a significant risk factor for developing cataracts in oncology patients.

The Impact of Oxygen Pulse and Its Curve Patterns on Male Patients with Heart Failure, Chronic Obstructive Pulmonary Disease, and Healthy Controls-Ejection Fractions, Related Factors and Outcomes.

Oxygen pulse (O2P) is a function of stroke volume and cellular oxygen extraction and O2P curve pattern (O2PCP) can provide continuous measurements of O2P. However, measurements of these two components are difficult during incremental maximum exercise. As cardiac function is evaluated using ejection fraction (EF) according to the guidelines and EF can be obtained using first-pass radionuclide ventriculography, the aim of this study was to investigate associations of O2P%predicted and O2PCP with EF in patients with heart failure with reduced or mildly reduced ejection fraction (HFrEF/HFmrEF) and chronic obstructive pulmonary disease (COPD), and also in normal controls. This was a prospective observational cross-sectional study. Correlations of resting left ventricular EF, dynamic right and left ventricular EFs and outcomes with O2P% and O2PCP across the three participant groups were analyzed. A total of 237 male subjects were screened and 90 were enrolled (27 with HFrEF/HFmrEF, 30 with COPD and 33 normal controls). O2P% and the proportions of the three types of O2PCP were similar across the three groups. O2P% reflected dynamic right and left ventricular EFs in the control and HFrEF/HFmrEF groups, but did not reflect resting left ventricular EF in all participants. O2PCP did not reflect resting or dynamic ventricular EFs in any of the subjects. A decrease in O2PCP was significantly related to nonfatal cardiac events in the HFrEF/HFmrEF group (log rank test, p = 0.01), whereas O2P% and O2PCP did not predict severe acute exacerbations of COPD. The findings of this study may clarify the utility of O2P and O2PCP, and may contribute to the currently used interpretation algorithm and the strategy for managing patients, especially those with HFrEF/HFmrEF. (Trial registration number NCT05189301.).

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains.

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.

Compressive strength of lithium disilicate inlay cementation on three different composite resins.

BACKGROUND/PURPOSE: Ceramic restorations have been increasingly applied over recent years. But the performance of cement is still unknown after cementation. This study was aimed to compare the compressive strength and the performances of three different types of composite resin after lithium disilicate inlay cementation. MATERIALS AND METHODS: Twenty-four human maxillary premolars were embedded in resin blocks, finished a MOD inlay preparation and scanned with an extraoral scanner. Lithium disilicate ceramic inlays (IPS e.max, Ivoclar Vivadent, Liechtenstein) were fabricated according to the scanner's model. All the specimens were then etched, bonded, and cemented with three different composite resins. Right after 5000 thermal cyclings, the specimens were accepted compressive tests to evaluate the compressive strength and failure types. Moreover, the fracture fragments of the specimens were examined using scanning electron microscopy (SEM) to verify the fracture type. RESULTS: Dual-cured resin cement (Rely X Ultimate) showed the highest compressive strength (1002 ±â€¯508 N), followed by the light-cured flowable resin (Z350 XT) (971 ±â€¯209 N) and light-cured bulkfill (Filtek Bulkfill) resin (581 ±â€¯191 N). Type IV (root fracture) failures in the dual-cured resin cement group was 25%, and light-cured flowable resin was 37.5%. But none of type IV fracture was found in the light-cured bulkfill flowable group. CONCLUSION: Dual-cured resin cement demonstrates the highest compressive strength after ceramic inlay cementation. Light-cured bulkfill resin shows the lowest compressive strength, but catastrophic failure is absent in this group.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Structural analyses of the PKA RIIß holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.