Search

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 andin situproximity 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.

Objectives: To investigate the association of the prognostic risk score CAPRA&PDE4D5/7/9 as measured on pre-surgical diagnostic needle biopsy tissue with pathological outcomes after radical prostatectomies in a clinically low–intermediate-risk patient cohort. Patients and Methods: RNA was extracted from biopsy punches of diagnostic needle biopsies. The patient cohort comprises n = 151 patients; of those n = 84 had low–intermediate clinical risk based on the CAPRA score and DRE clinical stage 2, or pathological pT stage > pT3a, or tumor penetrated prostate capsular status, or pN1 disease); (ii) any ISUP pathological Gleason >2; (iii) any ISUP pathological Gleason >1. In the n = 84 patients with low to intermediate clinical risk profiles, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk score was significantly lower in patients with favorable vs. unfavorable outcomes. In univariable logistic regression modeling the genomics PDE4D5/7/9_BCR as well as the clinical-genomics CAPRA&PDE4D5/7/9_BCR combination model were significantly associated with all three post-surgical pathology outcomes (p = 0.02, p = 0.0004, p = 0.04; and p = 0.01, p = 0.0002, p = 0.01, respectively). The clinically used PRIAS criteria for the selection of low-risk candidate patients for active surveillance (AS) were not significantly associated with any of the three tested post-operative pathology outcomes (p = 0.3, p = 0.1, p = 0.1, respectively). In multivariable analysis adjusted for the CAPRA score, the genomics PDE4D5/7/9_BCR risk score remained significant for the outcomes of adverse pathology (p = 0.04) and ISUP pathological Gleason >2 (p = 0.004). The negative predictive value of the CAPRA&PDE4D5/7/9_BCR risk score using the low-risk cut-off (0.1) for the three pathological endpoints was 82.0%, 100%, and 59.1%, respectively for a selected low-risk cohort of n = 22 patients (26.2% of the entire cohort) compared to 72.1%, 94.4%, and 55.6% for n = 18 low-risk patients (21.4% of the total cohort) selected based on the PRIAS inclusion criteria. Conclusion: In this study, we have shown that the previously reported clinical-genomics prostate cancer risk model CAPRA&PDE4D5/7/9_BCR which was developed to predict biological outcomes after surgery of primary prostate cancer is also significantly associated with post-surgical pathology outcomes. The risk score predicts adverse pathology independent of the clinical risk metrics. Compared to clinically used active surveillance inclusion criteria, the clinical-genomics CAPRA&PDE4D5/7/9_BCR risk model selects 22% (n = 8) more low-risk patients with higher negative predictive value to experience unfavorable post-operative pathology outcomes.

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.

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.

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.

International audience; Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

Objective: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR–TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. Conclusions: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1–37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

Significance Phosphodiesterase 10A (PDE10A) is as a target of interest in Huntington’s disease (HD) as levels of the enzyme have been shown to decrease prior to the development of the hallmark motor symptoms. Clearly, a better understanding of how PDE10A protein levels change as HD develops is required. Here we show that mutations in the regulatory GAF domains of PDE10A that cause hyperkinetic syndromes in humans lead to misprocessing of the PDE10A enzyme that ultimately leads to targeted degradation by the ubiquitin proteasome system or clearance by autophagy. Both mechanisms result in a paucity of PDE10A activity that lead to a loss of movement coordination. Our research suggests that similar mechanisms may underpin PDE10A loss during HD., A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington’s disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.

We demonstrated that Mas1 and ET B R physically interact in endothelial cells inducing Ang-(1-7) vascular protection. Using high throughput screening of >20,000 druggable compounds, we identified a number of molecules that enhance Mas1:ET B R interactions. Of these, 2 potently enhanced interaction between the receptors. These enhancers (Enh), termed Enh3 and Enh4 were used to assess Mas1:ET B R interaction and cellular functional responses in vascular smooth muscle cells (VSMCs) from normotensive WKY and hypertensive SHRSP rats. Cells were exposed to Enh 3 and Enh4 (10 -5 M) for short (5,15 and 30min) and long timepoints (5hours). Expression of signaling molecules was assessed by immunoblotting and Ca 2+ influx was evaluated using fluorescence microscopy. In WKY VSMCs, Enh 3 short term stimulation reduced basal ERK1/2 phosphorylation (26.8±5.9% vs veh, p2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. In conclusion, enhancing Mas1:ET B R interaction attenuates mitogenic and pro-inflammatory signaling pathways in WKY and SHRSP VSMCs. Enhancing interaction between these receptors does not increase Ca 2+ signaling, important in VSMC contraction. Our data suggest that Enh3 and Enh4 may have VSMC protective effects and that they do not amplify injurious signaling induced by ET-1/ETBR. These findings identify a potential new strategy in vasoprotection.

Phosphodiesterases (PDEs), enzymes that degrade 3′,5′-cyclic nucleotides, are being pursued as therapeutic targets for several diseases, including those affecting the nervous system, the cardiovascular system, fertility, immunity, cancer and metabolism. Clinical development programmes have focused exclusively on catalytic inhibition, which continues to be a strong focus of ongoing drug discovery efforts. However, emerging evidence supports novel strategies to therapeutically target PDE function, including enhancing catalytic activity, normalizing altered compartmentalization and modulating post-translational modifications, as well as the potential use of PDEs as disease biomarkers. Importantly, a more refined appreciation of the intramolecular mechanisms regulating PDE function and trafficking is emerging, making these pioneering drug discovery efforts tractable.

Background Dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) belongs to a family of CMGC kinases that function as modulators of different downstream pathways that allow cells to cope with hypoxia, DNA damage and various stress signals. Additionally, DYRK2 has been implicated in various human cancers with both pro- and anti-tumour roles, which are probably cancer type- and cell type-dependent. Furthermore, studies show that DYRK2 is involved in epithelial-mesenchymal transition, hence suggesting a role in tumour metastasis. The current study investigates the prognostic role of DYRK2 in breast cancer and investigates its potential as a novel therapeutic target. Methods Immunohistochemistry was employed to investigate if nuclear expression of DYRK2 was associated with clinical outcome measures in a cohort of 715 patients. Expression was determined using the weighted histoscore method. Antibody specificity was confirmed in paraffin embedded cell pellets +/- DYRK2 silencing. Cell counts in parental and CRISPR-mediated DYRK2 knocked-out MDA-MB-468 and MDA-MB-231 cells (ER, PR, HER2, AR negative) were measured using Alamar Blue; NSGTMmice (n=8) were injected subcutaneously with MDA-MDB-231 with or without DYRK2 depletion to assess tumour growth in vivo. Results In a cohort of 715 patients, median follow-up was 160 months with 155 breast cancer deaths and 135 deaths due to other causes. The majority of patients were over 50 years of age (71%), had ductal carcinoma (88%), tumours 145 were classified as high expression. In the full cohort (p=0.087) and ER negative (p=0.066) cohort DYRK2 was not associated with cancer specific survival. However in TN disease high DYRK2 expression was associated with cancer specific survival (p=0.012, mean survival 145 months versus 107 months). This was potentiated in patients with ER, PR, HER2, AR negative disease (p=0.005, mean survival 166 months versus 100 months) and independent in multivariate analysis with age, histological tumour type, tumour size tumour grad, nodal status, ki67 index, chemotherapy, radiotherapy and recurrence (p=0.13, HR 3.920). Following this observation, patients with ER, AR negative disease were investigated and again high DYRK2 expression was associated with cancer specific survival (p=0.0003, mean survival 163 months versus 86 months) and was independent when combined in multivariate analysis (p=0.001, HR 4.154). To investigate if DYRK2 was a potential target in TN breast cancer, the effect of silencing DYRK2 was investigated. CRISPR-mediated DYRK2 depletion impeded cell proliferation in TN cell-lines and markedly reduced tumour burden in mouse MDA-MDB-231 xenografts (p Conclusions Our studies indicate that DYRK2 is indeed a potential therapeutic target for patients with TN breast cancer or ER, AR negative breast cancer. Citation Format: Edwards J, Baillie G, Quinn J, Monreno R, Banerjee S, Tomkinson N, MacKay S, De La Vega L. DYRK2 is a novel therapeutic target in ER negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-10.

COVID‐19 is caused by a novel coronavirus called severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Virus cell entry is mediated through a protein‐protein interaction (PPI) between the SARS‐CoV‐2 spike protein and angiotensin‐converting enzyme 2 (ACE2). A series of stapled peptide ACE2 peptidomimetics based on the ACE2 interaction motif were designed to bind the coronavirus S‐protein RBD and inhibit binding to the human ACE2 receptor. The peptidomimetics were assessed for antiviral activity in an array of assays including a neutralization pseudovirus assay, immunofluorescence (IF) assay and in‐vitro fluorescence polarization (FP) assay. However, none of the peptidomimetics showed activity in these assays, suggesting that an enhanced binding interface is required to outcompete ACE2 for S‐protein RBD binding and prevent virus internalization.

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)–ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low μM range (Kd = 0.207–1.206 μM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, ‘miniproteins’, nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

Cardiac myosin binding protein-C (cMyBP-C) is a fundamental component of the cardiac sarcomere involved in regulating systolic and diastolic activity, processes which must be tightly maintained to preserve cardiac function. Importantly, as a non-enzymatic protein, cMyBP-C relies solely on post-translational modifications and protein-protein interactions in order to modulate its function, and does so through phosphorylation, glutathionylation and acetylation amongst others. Although some are better understood than others, these modifications may represent novel therapeutic routes to modulate cMyBP-C function in the treatment of cardiac disease.

Much interest has been expended lately in characterizing the association between DExH-Box helicase 9 (DHX9) dysregulation and malignant development, however, the enigmatic nature of DHX9 has caused conflict as to whether it regularly functions as an oncogene or tumor suppressor. The impact of DHX9 on malignancy appears to be cell-type specific, dependent upon the availability of binding partners and activation of inter-connected signaling pathways. Realization of DHX9’s pivotal role in the development of several hallmarks of cancer has boosted the enzyme's potential as a cancer biomarker and therapeutic target, opening up novel avenues for exploring DHX9 in precision medicine applications. Our review discusses the ascribed functions of DHX9 in cancer, explores its enigmatic nature and potential as an antineoplastic target., Lay abstract DExH-Box helicase 9 (DHX9) is an enzyme with multiple important functions in cells, therefore, its deregulated activity can cause alterations in cellular growth and subsequent formation of tumors. In particular, irregular DHX9 activity is attributed to the development of several hallmarks of cancer, however, it can have both pro- and anti-cancer effects, thus leading to conflicting views on DHX9's role in cancer development. The diverse implications of DHX9 in malignancy exposes the protein as a potential marker for cancer detection and intervention. Our review discusses the vast array of DHX9’s functions in cancer development, and its potential as a target for cancer treatment.

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.

Mas and ET B receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET B R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET B R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10 -7 M) in the presence/absence of A779 (Mas receptor antagonist, 10 -5 M) and BQ788 (ET B R antagonist, 10 -5 M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H 2 O 2 )). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET B R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (pB R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10 -5 M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p2 O 2 production (54.7±11.1% vs ctl, p2 O 2 generation (48.1±15.4% vs ctl, pNrf2 (3.0 fold), Sod1 (2.0 fold) and Nqo1 (3.1 fold) mRNA expression (pB R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET B R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.

The ability of inducible regulator suppressor of cytokine signaling 3 (SOCS3) to inhibit Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling requires interaction with specific cytokine receptors, JAKs, and components of the cellular ubiquitylation machinery. However, it is now clear that additional protein interactions are essential for effective inhibition of JAK-STAT signaling that have also identified new roles for SOCS3. For example, we have demonstrated that SOCS3 interaction with cavin-1, a core component of caveolae essential for their formation, is required for effective inhibition of interleukin (IL)-6 signaling and maintenance of cellular levels of caveolae. This is achieved through cavin-1 interaction with a discrete motif within the SOCS3 SH2 domain. Here, we describe in detail three methods (coimmunoprecipitation; peptide pull-down; peptide array overlay) we have used to validate and characterize cavin-1/SOCS3 interactions in vitro.

Phosphodiesterases (PDEs) are the only superfamily of enzymes that have the ability to break down cyclic nucleotides and, as such, they have a pivotal role in neurological disease and brain development. PDEs have a modular structure that allows targeting of individual isoforms to discrete brain locations and it is often the location of a PDE that shapes its cellular function. Many of the eleven different families of PDEs have been associated with specific diseases. However, we evaluate the evidence, which suggests the activity from a sub-family of the PDE4 family, namely PDE4B, underpins a range of important functions in the brain that positions the PDE4B enzymes as a therapeutic target for a diverse collection of indications, such as, schizophrenia, neuroinflammation, and cognitive function.

Fibrinolysis is an integral part of the matrix remodeling process that contributes to tissue repair. Fibrin clots are broken down during fibrinolysis in a controlled process. Fibrin degradation products (FDPs) have also been shown to have a role in the regulation of cell growth and are implicated in various vascular diseases. This protocol was designed to quantitatively measure the extent of fibrin breakdown and how this can be adapted as a tool to further investigate the pathway involved in fibrinolysis or fibrin degradation products. Until now, we haven't found an alternative method to analysis fibrinolysis.

The ability of inducible regulator suppressor of cytokine signaling 3 (SOCS3) to inhibit Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling requires interaction with specific cytokine receptors, JAKs, and components of the cellular ubiquitylation machinery. However, it is now clear that additional protein interactions are essential for effective inhibition of JAK-STAT signaling that have also identified new roles for SOCS3. For example, we have demonstrated that SOCS3 interaction with cavin-1, a core component of caveolae essential for their formation, is required for effective inhibition of interleukin (IL)-6 signaling and maintenance of cellular levels of caveolae. This is achieved through cavin-1 interaction with a discrete motif within the SOCS3 SH2 domain. Here, we describe in detail three methods (coimmunoprecipitation; peptide pull-down; peptide array overlay) we have used to validate and characterize cavin-1/SOCS3 interactions in vitro.

Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE activity plays a central role in disease pathogenesis. This highlights the need for developing methods that can be used to further understand PDE function and assess the effectiveness of potentially novel PDE therapeutics. Here we describe such an approach, where PDE activity is indirectly measured through the direct quantification of radioactively tagged cAMP (pmol/min(-1)/mg(-1)). This method provides a highly sensitive tool for investigating PDE functionality.

Phosphodiesterase (PDE) inhibitors are currently under evaluation as agents that may facilitate the improvement of cognitive impairment associated with Alzheimer's disease. Our aim was to determine whether inhibitors of PDEs 4, 5 and 9 could alleviate the cytotoxic effects of amyloid beta 1–42 (Aβ1–42) via a mechanism involving the small heatshock protein HSP20. We show that inhibition of PDEs 4, 5 and 9 but not 3 induces the phosphorylation of HSP20 which, in turn, increases the colocalisation between the chaperone and Aβ1–42 to significantly decrease the toxic effect of the peptide. We conclude that inhibition of PDE9 is most effective to combat Aβ1–42 cytotoxicity in our cell model.

Spatio-temporal regulation of localised cAMP nanodomains is highly dependent upon the compartmentalised activity of phosphodiesterase (PDE) cyclic nucleotide degrading enzymes. Strategically positioned PDE–protein complexes are pivotal to the homeostatic control of cAMP-effector protein activity that in turn orchestrate a wide range of cellular signalling cascades in a variety of cells and tissue types. Unsurprisingly, dysregulated PDE activity is central to the pathophysiology of many diseases warranting the need for effective therapies that target PDEs selectively. This short review focuses on the importance of activating compartmentalised cAMP signalling by displacing the PDE component of signalling complexes using cell-permeable peptide disrupters

Phosphodiesterases (PDEs) have long been considered as targets for the treatment of Alzheimer's disease (AD) and a substantial body of evidence suggests that one sub-family from the super-family of PDEs, namely PDE4D, has particular significance in this context. This review discusses the role of PDE4 in the orchestration of cAMP response element binding signaling in AD and outlines the benefits of targeting PDE4D specifically. We examine the limited available literature that suggests PDE4 expression does not change in AD brains together with reports that show PDE4 inhibition as an effective treatment in this age-related neurodegenerative disease. Actually, aging induces changes in PDE4 expression/activity in an isoform and brain-region specific manner that proposes a similar complexity in AD brains. Therefore, a more detailed account of AD-related alterations in cellular/tissue location and the activation status of PDE4 is required before novel therapies can be developed to target cAMP signaling in this disease.

Objectives. To investigate the added value of assessing transcripts for the long cAMP phosphodiesterase-4D (PDE4D) isoforms, PDE4D5 and PDE4D9, regarding the prognostic power of the ‘CAPRA & PDE4D7’ combination risk model to predict longitudinal postsurgical biological outcomes in prostate cancer.Patients and Methods. RNA was extracted from both biopsy punches of resected tumours (606 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). RT-qPCR was performed in order to determine PDE4D5, PDE4D7, and PDE4D9 transcript scores in both study cohorts. By RNA sequencing, we determined the TMPRSS2-ERG fusion status of each tumour sample in the RP cohort. Kaplan-Meier survival analyses were then applied to correlate the PDE4D5, PDE4D7 and PDE4D9 scores with postsurgical patient outcomes. Logistic regression was then used to combine the clinical CAPRA score with PDE4D5, PDE4D7, and PDE4D9 scores in order to build a ‘CAPRA & PDE4D5/7/9’ regression model. ROC and decision curve analysis was used to estimate the net benefit of the ‘CAPRA & PDE4D5/7/9’ risk model.Results. Kaplan-Meier survival analysis, on the RP cohort, revealed a significant association of the PDE4D7 score with postsurgical biochemical recurrence (BCR) in the presence of the TMPRSS2-ERG gene rearrangement (logrank pConclusion. Association of the long PDE4D5, PDE4D7, and PDE4D9 transcript scores to prostate cancer patient outcome, after primary intervention, varies in opposite directions depending on the TMPRSS2-ERG genomic fusion background of the tumour. Adding transcript scores for the long PDE4D isoforms, PDE4D5 and PDE4D9, to our previously presented combination risk model of the combined ‘CAPRA & PDE4D7’ score, in order to generate the CAPRA and PDE4D5/7/9 score, significantly improves the prognostic power of the model in predicting postsurgical biological outcomes in prostate cancer patients.

SummaryTo survive aneuploidy-induced proteotoxic stress, cancer cells activate the proteotoxic-stress response pathway, which is controlled by heat shock factor 1 (HSF1). This pathway supports cancer initiation, cancer progression and chemoresistance and thus is an attractive target. As developing HSF1 inhibitors is challenging, the identification and targeting of upstream regulators of HSF1 presents a tractable alternative strategy. Here we demonstrate that in triple negative breast cancer (TNBC) cells, the dual-specificity tyrosine-regulated kinase 2 (DYRK2) phosphorylates HSF1, promoting its nuclear stability and transcriptional activity. Thus, DYRK2 depletion reduces HSF1 activity and sensitises TNBC cells to proteotoxic stress. Importantly, in tumours from TNBC patients, DYRK2 levels positively correlate with active HSF1 and associates with poor prognosis, suggesting that DYRK2 could be promoting TNBC. In agreement with this, DYRK2 depletion reduces tumour growth in a TNBC xenograft model. These findings identify DYRK2 as both, a key modulator of the HSF1 transcriptional program, and a potential therapeutic target.

The many functions of β-arrestin proteins in the desensitization of G-protein-coupled receptors have been well characterized; however, the discovery that this scaffold protein could actually recruit phosphodiesterases (PDEs) to the site of cAMP synthesis changed the way researchers thought about the static nature of precisely localized cAMP hydrolysis by anchored PDEs. Before this discovery, the compartmentalization of cAMP gradients formed by the activation of specific receptors was generally understood to be underpinned by highly localized pools of specific PDEs that were anchored by large static anchors such as A-kinase-anchoring proteins (AKAPs). Such anchors acted to position cAMP effector proteins such as protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) in places that would allow cAMP concentrations to breach their activation threshold only when a specific receptor activation occurred. In this arrangement PDEs acted as local "sinks" for cAMP and this enforced receptor-specific function by allowing the correct activation of a distinct pool of cAMP effectors in precise localizations. The discovery that β-arrestin could shuttle cAMP hydrolyzing activity to the membrane shortly after receptor activation added to the complexity of this process by restricting cAMP diffusion into the cell interior for some receptors. This chapter describes the methods used to identify, confirm, and test the function of PDE-β-arrestin complexes.

The dual-specific cAMP/cGMP phosphodiesterase PDE10A is exclusively localised to regions of the brain and specific cell types that control crucial brain circuits and behaviours. The downside to this expression pattern is that PDE10A is also positioned to be a key player in pathology when its function is perturbed. The last decade of research has seen a clear role emerge for PDE10A inhibition in modifying behaviours in animal models of psychosis and Huntington's disease. Unfortunately, this has not translated to the human diseases as expected. More recently, a series of families with hyperkinetic movement disorders have been identified with mutations altering the PDE10A protein sequence. As these mutations have been analysed and characterised in other model systems, we are beginning to learn more about PDE10A function and perhaps catch a glimpse into how PDE10A activity could be modified for therapeutic benefit.

The many functions of β-arrestin proteins in the desensitization of G-protein-coupled receptors have been well characterized; however, the discovery that this scaffold protein could actually recruit phosphodiesterases (PDEs) to the site of cAMP synthesis changed the way researchers thought about the static nature of precisely localized cAMP hydrolysis by anchored PDEs. Before this discovery, the compartmentalization of cAMP gradients formed by the activation of specific receptors was generally understood to be underpinned by highly localized pools of specific PDEs that were anchored by large static anchors such as A-kinase-anchoring proteins (AKAPs). Such anchors acted to position cAMP effector proteins such as protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) in places that would allow cAMP concentrations to breach their activation threshold only when a specific receptor activation occurred. In this arrangement PDEs acted as local "sinks" for cAMP and this enforced receptor-specific function by allowing the correct activation of a distinct pool of cAMP effectors in precise localizations. The discovery that β-arrestin could shuttle cAMP hydrolyzing activity to the membrane shortly after receptor activation added to the complexity of this process by restricting cAMP diffusion into the cell interior for some receptors. This chapter describes the methods used to identify, confirm, and test the function of PDE-β-arrestin complexes.