Regulation of the PP2AC, PP4C, PP6C and alpha4 signalling axis in the myocardium : roles in calcium homeostasis and hypertrophy

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of most proteins, which is controlled by the opposing reactions of protein kinases and phosphatases (PP). The type 2A protein phosphatase family is comprised of PP2A, PP4 and PP6, due to the high amino acid homology of their catalytic subunits (PP2ACα/β, PP4C and PP6C). The activity and expression of this family are partly regulated by alpha4, a common regulatory protein that is essential in type 2A phosphatase holoenzyme biogenesis. In the heart, more than 98% of protein dephosphorylation is mediated by serine/ threonine protein phosphatases, of which type 2A protein phosphatases along with protein phosphatase 1, contr ibute approximately 90%. Currently, the role(s) of type 2A protein phosphatases and their regulation by alpha4 in the heart is poorly defined and requires detailed investigation. In this study, quantitative PCR analysi s demonstrated that PP2ACβ mRNA was most abundant in H9c2 cardiomyocytes and neonatal rat ventricular myocytes (NRVM) whilst, in adult rat ventricular myocytes (ARVM), PP2ACα mRNA was the most abundantly transcribed. Surprisingly, immunoblotting analysis, using catalytic subunit-specific antibodies, identified the expression of all type 2A protein phosphatase catalytic subunits in H9c2 cardiomyocytes and NRVM, however, ARVM only expressed PP2AC and PP6C protein. PP4C protein expression was only detectable in ARVM following proteasomal inhibition with compound MG132. Using siRNA to selectively knockdown type 2A protein phosphatase catalytic subunits, it was revealed that PP2ACα alone dephosphorylates CaV1.2-Ser1928. The data also suggested that PP2ACα, PP2ACβ and PP4C dephosphorylate phospholemman at both Ser63 and Ser68 in cardiomyocytes. siRNA-mediated knockdown of alpha4 protein expression rapidly reduced the expression of all type 2A catalytic subunits. Interestingly, expression of both PP2AC and alpha4 protein expression was elevated in pressure overload-induced left ventricular (LV) hypertrophy. Even though PP6C expression was unchanged, expression of PP6C regulatory subunits (i) SIT4-associated protein 1 (SAP1) and (ii) ankyrin repeat domain (ANKRD) 28 and 44 proteins were upregulated, whereas SAP2 expression was downregulated in hypertrophied LV tissue. Co-immunoprecipitation experiments revealed that the cellular association between alpha4 protein and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Exposure of cardiomyocytes to hydrogen peroxide increased levels of H2AX phosphorylation (γH2AX), indicating hydrogen peroxide-induced DNA damage, which was unaffected by the knockdown of PP6C, however, levels of both total H2AX and γH2AX were diminished by the knockdown of alpha4 protein. The novel findings in this study collectively, demonstrate the differences in th e expression, stability, substrate specificity and altered alpha4-mediated regulation of the type 2A protein phosphatases in normal and hypertrophied myocardium and provide new insights into the molecular mechanisms involved in cardiac calcium homeostasis and DNA repair and thereby help to identify potential targets for the development of new and improved therapies against cardiac pathological hypertrophy

Genome Wide CRISPR/Cas9 Screen Identifies the Coagulation Factor IX (F9) as a Regulator of Senescence

[Abstract] During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.This paper was funded by the BBSRC (BB/P000223/1), the MRC (MR/K501372/1), The Royal Society (RG170399) and Barts Charity (MGU0497 and G-002158) grants to A.O. M.M. was funded by PI19/00145, IN607B2020/12 and 858014. J.F.L. is funded by Xunta de Galicia (ED481B 2017/117). M.B. was funded by MRC (MR/K501372/1). T.P.M. was funded by a QMUL PhD programme and T.D.N.’s lab is currently funded by a Barts Charity project grant (MGU0534). P.C.F. is currently funded by GAIN (IN606C 2021/006) Xunta de GaliciaReino Unido. Biotechnology and Biological Sciences Research Council; BB/P000223/1Reino Unido. Medical Research Council; MR/K501372/1Reino Unido. Royal Society; RG170399Barts Charity (Londres); MGU0497Barts Charity (Londres); G-002158Xunta de Galicia; IN607B2020/12Xunta de Galicia; ED481B 2017/117Barts Charity (Londres); MGU0534Xunta de Galicia; IN606C 2021/00

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Research Letter: Mechanism-based inhibition of HsaD: a C-C bond hydrolase essential for survival of M. tuberculosis in macrophage

M. tuberculosis remains the leading cause of death by a bacterial pathogen worldwide. Increasing prevalence of multidrug resistant organisms means prioritising identification of targets for anti-tuberculars. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD), part of the cholesterol metabolism operon, is vital for survival within macrophage. The C-C-bond hydrolase, HsaD has a serine protease-like catalytic triad. We tested a range of serine protease and esterase inhibitors for their effects on HsaD activity. As well as providing a potential starting point for drug development, the data provides evidence for the mechanism of C-C bond hydrolysis. This screen also provides a route to initiate development of fragment based inhibitors. This article is protected by copyright. All rights reserved

Lipidomics Analysis of Free Fatty Acids in Human Plasma of Healthy and Diabetic Subjects by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

Targeted analytical methods for the determination of free fatty acids (FFAs) in human plasma are of high interest because they may help in identifying biomarkers for diseases and in monitoring the progress of a disease. The determination of FFAs is of particular importance in the case of metabolic disorders because FFAs have been associated with diabetes. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of 74 FFAs in human plasma. The method is fast (10-min run) and straightforward, avoiding any derivatization step and tedious sample preparation. A total of 35 standard saturated and unsaturated FFAs, as well as 39 oxygenated (either hydroxy or oxo) saturated FFAs, were simultaneously detected and quantified in plasma samples from 29 subjects with type 2 diabetes mellitus (T2D), 14 with type 1 diabetes mellitus (T1D), and 28 healthy subjects. Alterations in the levels of medium-chain FFAs (C6:0 to C10:0) were observed between the control group and T2D and T1D patients

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.We are grateful to Tom Nightingale and Maria Niklison-Chirou for reading the manuscript. Alissa Weaver provided tagged CD63 constructs; and Jacob Yount and I-Chueh Huang supplied the IFITM3 and shIFITM3 plasmids. We are grateful to Luke Gammon, the Queen Mary University of London (QMUL) Genome Centre, and Gary Warnes for excellent technical support. Mouse hepatic stellate cells were a gift from Scott Lowe. A.O.’s lab is supported by the BBSRC (BB/P000223/1) and The Royal Society(RG170399). M.B. is funded by the MRC (MR/K501372/1) and the Centre for Genomics and Child Health. P.C.-F. (IN606B 2017/014) and J.F.-L.(ED481B 2017/117) are funded by the Xunta de Galicia.S