Investigating the human myometrial acetylome and identifying potential new targets for treating preterm labour

Preterm birth occurs in >50,000 cases per annum in the UK and is still the major cause of neonatal mortality and morbidity in the UK. Preterm babies that survive do so with an elevated risk of long-term physical or mental handicap. Globally, the WHO estimate that up to 15 million births per annum occur prematurely with approximately 1 million neonatal deaths. The development of better therapeutics in managing preterm birth, therefore, is a prime research aim that serves to tackle the considerable health and socio-economic impacts of this disorder. A major contributor to the incidence of preterm birth is premature activation of uterine contractions, even in the absence of other clinical risk associations such as infection, yet there still is no broadly efficient therapeutic treatment free of risk factors for the mother and/or neonate. At present, our understanding of the mechanisms that control activation of the uterus at term or preterm is weak compared to other organ systems and this is reflected in our inability to prescribe drugs that limit premature uterine contractility without jeopardising neonatal outcome. This project seeks to address these issues.
Recent technical and experimental advances in high throughput proteomics have made us aware of an expanding range of protein modifications that may regulate cell function. Published evidence from our laboratory has indicated that myofilament-associated protein acetylation is an important mechanism for regulating human myometrial contractility. Drugs which act to increase protein acetylation by inhibiting lysine deacetylases (KDACs) not only reduce human myometrial contractility in vitro but also delay uterine activation in an animal model of preterm labour whilst improving neonatal survival. In the present study we will employ advanced proteomic techniques to identify proteins that are targets for acetylation in these processes. Utilising a cross-disciplinary approach, encompassing scentific and clinical expertise, and experimentation on human ex vivo biopsies in alliance with in vivo animal model of preterm labour, we will provide novel identification of changes in myometrial protein acetylation, including the identification of individual acetylation sites, with treatment by KDAC inhibition or lysine acetylase (KAT) activation. In concert, we will establish the ability of KDAC inhibition or KAT activation to delay experimentally-induced preterm labour and improve neonatal survival and outcome. As these are the defining aspirations of developing any new tocolytic drugs, our data will not only inform us of novel molecular mechanisms involved in regulating term and preterm labour, but will also inform us of promising protein targets for development of future therapeutics for preterm labour. Beyond the immediate scientific and clinical research practitioners interested in pregnancy and pregnancy complications, this work will also provide a publicly-available dataset with a broad scale assessment of human and guinea pig cell protein acetylation which will be of interest to comparative biologists and proteomic bioinformaticians.

Data from our group indicate the importance of myofilament-associated protein acetylation in regulating human myometrial contractility. A multi-faceted strategy will be followed to determine the extent of myometrial acetylated proteins utilising human biopsies from pregnant women at term and tissues obtained from a clinically relevant animal model of human pregnancy/preterm labour. Our aim is to identify myometrial cell proteins that change their acetylation status by pharmacological manipulation. These may thus serve as potential novel targets for the future development of agents to treat preterm labour.
The Stable Isotope Labelling of Cells in Culture (SILAC) of primary myometrial cells treated to increase protein acetylation with the lysine deacetylase (KDAC) inhibitor TSA or lysine acetyltransferase (KAT) agonist CTB, will be established. Protein lysates from these cultures will be trypsin digested and acetylated peptides immunoprecipitated using lysine acetylated-specific antibodies and subsequently fractionated using an Agilent 3100 OffGel fractionater and analysed using LCMSMS on a Dionex U3000 nano-HPLC system coupled to an Orbitrap LTQ XL Discovery mass spectrometer. Peptides will be distinguished by their unique mass/charge ratios that will enable (i) identification of any newly acetylated peptide sequences and (ii) assessment of the relative changes in abundance of acetylated species. The peptides from SILAC will than serve as internal standards for MS analysis of the protein acetylation changes in the native human tissues at rest or treated with TSA/CTB. Our preliminary data also suggest a potential tocolytic benefit of TSA treatment in the Guinea pig model of preterm labour. Similar proteomic procedures will, therefore, be performed in myometrial tissues from animals undergoing preterm labour both before and after treatment with TSA/CTB. The neonatal outcome will be subsequently established by following pups for 3 months post-natally.

The impact of this research will be to elucidate novel molecular and cellular mechanisms that control uterine activity during term and preterm labour and, in so doing, identify potential targets for the development of new tocolytic drug treatments.

In the developed world, premature birth complicates 6-10% of pregnancies and the incidence of birth before 28 weeks gestation (extreme prematurity) is increasing: those infants having elevated risks of major long-term mental and physical handicap. Moreover, such infants have a disproportionate effect on health-care budgets world-wide. In this context a recent estimate of the total cost (England and Wales) of caring for preterm newborns to adulthood to the public sector was £2.95 billion per annum. Current tocolytic therapies have limited use and are associated with complications for both infant and mother. Since the antenatal health of a baby is a major predictor of adult morbidity, reducing the incidence of premature birth is an unequivocal healthcare goal. To do this, it is necessary to increase our knowledge base of the molecular and cellular processes modulating uterine activity in order to inform the design of better therapeutics for targeting premature contractions prior to term. This approach offers promise for, in the long term, impacting on the socio-economic and health burdens associated with preterm labour. Our work is designed to address these issues. Using a cross-disciplinary approach at the scientific-clinical interface, it will elucidate, for the first time, the role of protein acetylation in controlling uterine function at term and during preterm labour. High sensitivity proteomic approaches will identify acetylated proteins, and the acetylation sites, involved in these processes and, in addition, assess the ability of pharmacological agents that interfere with this process to ameliorate preterm labour and improve neonatal outcome.

The research will, therefore be of interest to academic and non-academic parties with wide-ranging interests including:
(i) scientists and clinical academics with an interest in the mechanisms of parturition and preterm labour; clinicians and commercial companies with a particular interest in the development of new approaches towards tocolytic tretatment of women at risk of preterm labour; opinion-formers and policy makers actively promoting the need for new research approaches to tackle the global health and socio-economic impact of preterm birth (e.g. the World Health Organisation:
http://www.who.int/pmnch/media/news/2012/201204_borntoosoon-report.pdf).
(ii) scientists interested in the regulation of mammalian cell biology by protein post-translational modification in general and acetylation in particular; this includes not only biologists but also chemists, mass spectrometry specialists and bioinformaticians involved in proteome research.
(iii) comparative biologists with an interest in use of appropriate animal models of human disease; our comparisons of the molecular mechanisms of guinea pig and human pregnancy will be of interest to those engaging with the National Centre for the Replacement, Refinement and reduction of Animals in Research.
(iv) patients who have consented to provide biopsies for the research in particular and patient groups, in general, with an interest in pregnancy and pregnancy complications.