Biology subjects

MacDonald, J. A.

Publications and source records attributed to MacDonald, J. A..

9 recordsLinked to original sources

bioRxiv2026

Mapping the reactive cysteine landscape of the NOD-like receptor pyrin domain-containing 3 protein

NOD-like receptor pyring-containing protein 3 (NLRP3) is a cytosolic pattern recognition receptor that drives assembly of an oligomeric inflammasome complex to stimulate pro-inflammatory cytokine production and pyroptotic cell death. Extensive research has shown that NLRP3 activation is dependent on intracellular reactive oxygen species (ROS). Despite this, the precise targets of ROS in this process remain undefined. In this study, a combination of chemoproteomics, in vitro biochemical assays and molecular dynamics simulations were used to characterize reactive Cys and how they may impact NLRP3 conformation and activity status. Affinity-captured NLRP3 was subjected to reverse-thiol trapping to reveal a pool of reactive Cys residues that were identified in subsequent chemoproteomic analyses. C150, C196, and C884 were consistently detected with both in vitro and in cellulo experiments. Several NLRP3 mutants were generated; C150A and C196A (NACHT domain) as well as C933A and C941A (LRR domain) reduced IL-1b output in a reconstituted inflammasome assay. Molecular simulation of Cys sulfenylation in the LRR suggests structural alterations that reposition the trLRR loop to destabilize the inactive NLRP3 structure. Structural modelling provided insights for how Cys oxidation of the LRR may impact on NLRP3 structure. Collectively, the results establish a mechanistic understanding for how ROS may promote NLRP3 inflammasome activation.

biochemistry↗

bioRxiv2025

Molecular dynamics simulations reveal the impact of Ser295 phosphorylation on the structure of pyrin domain-containing NOD-like receptor 3

The nucleotide-binding, leucine rich repeat, and pyrin-containing 3 (NLRP3) protein is regulated by phosphorylation of Ser295 in the NACHT domain. This post-translational modification is known to inhibit the enzymatic ATPase activity of NLRP3 and impede inflammasome complex assembly. In this study, modeled structures of unphosphorylated and pSer295-phosphorylated NLRP3-{Delta}PYD were subjected to molecular dynamics simulations. The outputs showed Ser295 phosphorylation to induce topologic distention of subdomains that comprise the NACHT domain. The relative orientation of important residues within the nucleotide-binding domain (NBD) were altered. Notable structural changes were observed for important residues within the Walker B motif that immediately follow pSer295. A favorable electrostatic environment was created for two residues (Lys232 and His522) that interact with ADP. Several other basic residues could establish favourable charge-charge interactions with the dianionic phosphate of pSer295. Arg296 and Glu343 underwent a functional change from negative/stabilizing to positive/destabilizing interaction upon phosphorylation of Ser295. Taken together, the results suggest that local structural transformations within the NBD could have consequences on the catalytic efficiency of the enzyme and suppress nucleotide turnover.

biochemistry↗

bioRxiv2024

Cooperative involvement of zipper-interacting protein kinase (ZIPK) and the dual-specificity cell division cycle 14A phosphatase (CDC14A) in vascular smooth muscle cell migration

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal adhesion assembly dynamics. ZIPK silencing can induce cytoskeletal remodeling with disassembly of actin stress fiber networks and coincident loss of focal adhesion kinase (FAK)-pY397 phosphorylation. The link between ZIPK inhibition and FAK phosphorylation is unknown, and critical interactor(s) and regulator(s) are not yet defined. In this study, we further analyzed the ZIPK-FAK relationship in VSMCs. The application of HS38, a selective ZIPK inhibitor, to coronary artery vascular smooth muscle cells (CASMCs) suppressed cell migration, myosin light chain phosphorylation (pT18&pS19) and FAK-pY397 phosphorylation as well. This was associated with the translocation of cytoplasmic FAK to the nucleus. ZIPK inhibition with HS38 was consistently found to suppress the activation of FAK and attenuate the phosphorylation of other focal adhesion protein components (i.e., pCas130, paxillin, ERK). In addition, our study showed a decrease in human cell-division cycle 14A phosphatase (CDC14A) levels with ZIPK-siRNA treatment and increased CDC14A with transient transfection of ZIPK. Proximity ligation assays (PLA) revealed CDC14A localized with ZIPK and FAK. Silencing CDC14A showed an increase of FAK-pY397 phosphorylation. Ultimately, the data presented herein strongly support a regulatory mechanism of FAK in CASMCs by a ZIPK-CDC14A partnership; ZIPK may act as a key signal integrator to control CDC14A and FAK during VSMC migration.

biochemistry↗

bioRxiv2023

Sex dimorphism in the aged metabolic phenotype of smoothelin-like 1 (SMTNL1) deficient mice

Smoothelin-like 1 (SMTNL1) is expressed in smooth and skeletal muscle tissues as well as a variety of steroid hormone-sensitive tissues. SMTNL1 can play a sex-dependent regulatory role in skeletal muscle metabolism in mice. Previous studies have documented appreciable changes in muscle morphology and metabolic function of young male mice with genetic deletion of Smtnl1. SMTNL1 can also impact the energy metabolism and insulin sensitivity of female mice during pregnancy. Therefore, we investigated the metabolic outcome of global SMTNL1 knockout (KO) in male and female mice with advancing age using a comprehensive lab animal monitoring system (CLAMS). With ageing, body weight gain was markedly higher with a concomitant increase in whole body adiposity as well as specific white adipose depots in the absence of SMTNL1. Moreover, this genotypic difference in whole body adiposity was greater in the female cohort. The deletion of SMTNL1 was also associated with delayed satiety in mice fed a high fat diet, which was more pronounced in the female mice. A significant genotypic difference was also revealed for the metabolic energy balance in 12 month old animals of both sexes. The KO animals were metabolically less efficient and displayed a preference for carbohydrate catabolism. However, reduced glucose tolerance was observed only in the female group with the deletion of SMTNL1. Taken together, the current findings establish a novel role for SMTNL1 in modulating adiposity and energy metabolism with ageing in a sex dimorphic way.

biochemistry↗

bioRxiv2023

Structural Insights into the ATP-dependent Activation of NOD-like Receptor with Pyrin 3 (NLRP3) Protein by Molecular Dynamics Simulation

The inflammasome-forming NOD-like receptor containing pyrin-3 (NLRP3) protein is a critical player in the innate immune responses to cellular danger signals. New structural data of NLRP3 provide a framework to probe the conformational impact of nucleotide binding. In this study, microsecond molecular dynamics (MD) simulations were used to detail information on the unique structural conformations adopted by NLRP3 with ATP or ADP binding. Sampling convergence reflected a high degree of confidence in the MD simulations as shown by RMSD and protein-nucleotide concordance, favourable overall MM-PBSA ligand binding energies for both nucleotides and low cosine coefficients of the principal eigenvectors obtained with essential dynamics (ED) analysis. NLRP3-ADP simulations provide relatively stable conformations with few global rearrangements as shown by decreased protein RMSD, Rg, SASA, and solvent accessibility for the ADP-bound structure. In contrast, ATP binding induced increased flexibility and resulted in substantive conformational changes to the NLRP3 structure. Binding of ATP was thermodynamically favourable as shown by the {Delta}Gsolv and MM-PBSA calculations of complex free energies, and these NLRP3-ATP simulations resulted in similar structural transitions as observed in the activated NLRC4 empirical structure. Lastly, the active conformation of NLRP3 critically depends on hinging between the HD2 and LRR domains, whereby ATP binding drives local conformational changes that are conveyed to the global structure.

biochemistry↗

bioRxiv2022

Death-associated protein kinase 3 (DAPK3) regulates the myogenic reactivity of cerebral arterioles

The vascular smooth muscle (VSM) of resistance blood vessels displays intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic responses of cerebral arterioles are critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arterioles. DAPK3 is a Ser/Thr kinase involved in Ca2+- sensitization mechanisms of VSM contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. Finally, a slow return of myogenic tone was observed during the sustained exposure of cerebral arterioles to a suite of DAPK3 inhibitors. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signaling compensation in response to attenuation of DAPK3 activity. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

physiology↗

bioRxiv2021

Death-associated protein kinase 3 (DAPK3) contributes to intestinal epithelial wound healing and the resolution of experimental colitis in mice

Various signaling molecules affecting epithelial restitution and wound healing are dysregulated in ulcerative colitis. Recent evidence demonstrates the necessity of Hippo-YAP/TAZ signaling, interceded by cytoskeletal remodeling, for intestinal regeneration. Death-associated protein kinase 3 (DAPK3) is a regulator of actin cytoskeleton reorganization that controls proliferation and apoptosis. Pharmacological inhibition of DAPK3 in Caco-2 human intestinal epithelial cells (IECs) with the HS38 compound augmented cell proliferation and enhanced wound closure. This phenotype corresponded with the increased colocalization of Yes-associated protein (YAP) with F-actin, which is indicative of YAP activation. The administration of HS38 impeded the resolution of intestinal injury and attenuated epithelial-specific proliferation after acute colitis induced by dextran-sodium-sulphate (DSS) in mice. During recovery from DSS-induced colitis, IEC proliferation was repressed, and mice exhibited increased disease severity when HS38 was applied to inhibit DAPK3. Moreover, HS38 treatment increased YAP nuclear localization in IECs, an indicator of signal activation. In summary, this study established DAPK3 as a key factor in intestinal epithelial regeneration and colitis progression by way of YAP signaling. Nevertheless, the role that DAPK3 play in different cell types will need further investigation to decipher the full consequence of DAPK3 inhibition on epithelial homeostasis.

pharmacology and toxicology↗

bioRxiv2021

Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4 that occurs in response to osmotically-driven changes in cell swelling in the central nervous system. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of synthetic and recombinant AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD peptide of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 M. CaM bound two C-terminal AQP4 peptides with interactions observed for both the C- and N-lobes of CaM (Kd1: 3.6 M, Kd2: 113.6 M, respectively). A recombinant AQP4 protein domain (rAQP4ct, containing the entire cytosolic C-terminal domain sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 M. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data suggest that this unique adapter protein binding mode of CaM and AQP4 may be an important regulatory mechanism for the vesicular trafficking of AQP4.

biochemistry↗

bioRxiv2020

Enhanced diastolic dysfunction but preserved systolic function after acute pressure overload in the absence of smoothelin-like 1 protein

AimsSmoothelin-like 1 (SMTNL1), a protein kinase A/G target protein, modulates the activity and expression of myosin light chain phosphatase and thus plays an important role in regulating vasoconstriction. Increased myogenic reactivity of resistance arterioles is associated with SMTNL1 silencing, and elevated baseline vascular tone is increasingly recognized as a risk factor for development of hypertension and chronic congestive heart failure. Hence, in this study we assessed cardiac function in SMTNL1 knockout mice with and without accompanying acute cardiac stress (i.e., pressure overload by transverse aortic constriction). Methods and ResultsMale and female, global Smtnl1 knockout (KO) & wild-type (WT) mice were assessed at 10 weeks of age by echocardiography and electrocardiography to define baseline cardiac function. Gross dissection revealed distinct cardiac morphology only in male mice; hearts from KO animals were significantly smaller than WT littermates but the proportion of heart mass taken up by LV was greater. Non-invasive analyses of KO mice showed reduced resting heart rate with improved ejection fraction and fractional shortening as well as elevated aortic and pulmonary flow velocities relative to their WT counterparts, but only in the male cohort. We further investigated the impact of acute pressure overload on cardiac morphometry and hemodynamics in the absence of SMTNL1 in male cohort using echocardiography and pressure-volume (PV) loop measurements. Interestingly, PV loop analysis revealed diastolic dysfunction with significantly increased end diastolic pressure and LV relaxation time along with a steeper end diastolic pressure-volume relationship an indicator of stiffer heart, in the KO group when compared to WT Sham-operated group. Sham KO mice also showed elevated arterial elastance and total peripheral resistance. With acute pressure overload, systolic function was preserved, but diastolic dysfunction was exacerbated in KO mice with higher E/E ratio and myocardial performance index along with a prolonged isovolumetric relaxation time relative to the aortic-banded WT group. ConclusionTaken together, the findings support a novel, sex-dimorphic role for SMTNL1 in modulating cardiac structure and diastolic function. Significantly, impairment of diastolic function following pressure overload in young animals lacking SMTNL1 is mainly driven by increased systemic vascular resistance, which mimics the clinical pathophysiology of heart failure with preserved ejection fraction (HFpEF). Translational PerspectiveHeart failure with preserved ejection fraction (HFpEF) is characterized by the impairment of diastolic function and accounts for half of all heart failure cases. Unfortunately, there is as yet no proven therapy available for these patients as the pathophysiology is complicated with the presence of multiple comorbidities, microvascular dysfunction and a lack of an ideal animal model. The phenotype of Smtnl1 global deletion male mice exhibits intriguing similarities to HFpEF, with elevated microvascular resistance driving diastolic dysfunction and LV remodeling. As such the SMTNL1 KO mouse represents a novel pre-clinical model to study the molecular etiology of HFpEF.

physiology↗