Biology subjects

Baillie, G. S.

Publications and source records attributed to Baillie, G. S..

6 recordsLinked to original sources

bioRxiv2023

MYPT1 is a non-canonical AKAP that tethers PKA to the MLCP signaling node

The activity of myosin light chain phosphatase (MLCP) is fine-tuned by the phosphorylation status of the MLCP target subunit 1 (MYPT1), which is determined by the antagonistic effects of Rho kinase (ROCK) and cAMP/cGMP-dependent protein kinases (PKA and PKG). PKA is composed of two regulatory (PKA-R, of which four variants exist) and two catalytic (PKAcat) subunits. PKA is targeted to the vicinity of its substrates by binding to A kinase anchoring proteins (AKAPs). MYPT1 is part of a complex signaling node that includes kinases and other enzymes involved in signal transduction. We hypothesized that MYPT1 might function as an AKAP to target PKA to the MLCP signaling node. Using a combination of immunoprecipitation, affinity pulldown and in situ proximity ligation assay (PLA) in human platelets and endothelial cells, we show that MYPT1 directly interacts with all four PKA-R variants and mapped the interaction to a 200 residues long central region of MYPT1. The interaction does not involve the docking and dimerization domain of PKA-R typically required for binding to AKAPs. Using peptide array overlay we identified K595, E676 and the PKA/ROCK kinase substrate motif R693/R694/S695/T696 as critical for the interaction. Substitution of S695, T696 or both by aspartic acid or the corresponding phosphorylated residue abolished binding. Our findings reveal that MYPT1 functions as a non-canonical AKAP to anchor PKA to the vicinity of non-phosphorylated S695/T696, where PKA-R would prevent PKAcat, and potentially also ROCK, from interacting with and phosphorylating MYPT1.

cell biology↗

bioRxiv2022

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the proliferation and invasiveness of melanoma. MITF-dependent gene expression requires recruitment of transcriptional co-activators such as CBP/p300, but details of this process are not fully defined. Here, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. A combination of pulldown assays and nuclear magnetic resonance spectroscopy determined that MITF binds both TAZ1 and TAZ2 domains of CBP/p300 with high affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining relatively dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These results provide mechanistic insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

biochemistry↗

bioRxiv2022

zDHHC5 expression is increased in cardiac hypertrophy and reduced in heart failure but this does not correlate with changes in substrate palmitoylation

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodiumcalcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Information is lacking about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression was rapidly elevated during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes was not sufficient to drive changes in palmitoylation of zDHHC5 substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its Cterminal tail, and we found the palmitoylation of zDHHC5 may be increased in heart failure in the same manner as NCX1, suggesting additional regulatory mechanisms such as acyl-CoA availability may be involved. Importantly, this study provides the first evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

biochemistry↗

bioRxiv2022

Cardiac myosin binding protein-C palmitoylation is associated with increased myofilament affinity, reduced myofilament Ca2+ sensitivity and is increased in ischaemic heart failure.

Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of cardiac contractility through its interactions with the thick and thin filament. cMyBP-C is heavily influenced by post-translational modifications, including phosphorylation which improves cardiac inotropy and lusitropy, and S-glutathionylation, which impairs phosphorylation and is increased in heart failure. Palmitoylation is an essential cysteine modification that regulates the activity of cardiac ion channels and soluble proteins, however, its relevance to myofilament proteins has not been investigated. In the present study, we purified palmitoylated proteins from ventricular cardiomyocytes and identified that cardiac actin, myosin and cMyBP-C are palmitoylated. The palmitoylated form of cMyBP-C was more resistant to salt extraction from the myofilament lattice than the non-palmitoylated form. Isometric tension measurements suggest c-MyBP-C palmitoylation reduces myofilament Ca2+ sensitivity, with no change to maximum force or passive tension. Importantly, cMyBP-C palmitoylation levels are reduced at the site of injury in a rabbit model of heart failure but increased in samples from patients with ischaemic heart failure. Identification of cMyBP-C palmitoylation site revealed S-glutathionylated cysteines C635 and C651 are required for cMyBP-C palmitoylation, suggesting an interplay between the modifications at these sites. We conclude that structural and contractile proteins within the myofilament lattice are palmitoylated, with important functional consequences for cardiac contractile performance.

cell biology↗

bioRxiv2022

Reduced PDE4D7 expression in prostate cancer correlates with down-regulation of genomic elements within the upstream PDE4D coding region on chromosome 5

PDE4D7 expression is diminished during progression of PCa and the phosphodiesterase has been proposed as a prognostic biomarker. RNA sequencing of PCa tissue identified sequences in the PDE4D coding region on Chr5q12 exhibiting similarities in mRNA expression profile to PDE4D7. As previously identified, PART1 had a significantly similar expression pattern to PDE4D7 across samples. However, other genes also matched expression to PDE4D7, including miRNAs and lncRNAs. These novel PDE4D7 associated genes represent putative PCa biomarkers and could have mechanistic roles in PCa progression.

cancer biology↗

bioRxiv2020

Phosphodiesterase Type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that Popeye proteins exist in complexes with members of the PDE4 family in cardiac myocytes thus restricting cAMP signaling. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 is essential to maintain action potential duration in beating cardiac myocytes.

biochemistry↗