Characterising the palmitoylation and SUMOylation of cardiac myosin binding protein-C in cardiac health and disease

Cardiac myosin binding protein-C (cMyBP-C) is a 12-domain sarcomeric accessory protein that transiently interact with actin, tropomyosin and myosin and regulates the activity of the myofilament to maintain systolic and diastolic function. cMyBPC is influenced by an increasing list of post-translational modifications (PTMs), including phosphorylation, which occurs predominantly in the N-terminal regions and regulates myofilament force and calcium sensitivity. Whilst the central domains have remained lesser studied, evidence suggests they are may promote different conformations of cMyBP-C, influence myosin binding and are a hot-spot for PTMs. This includes the cysteine modification S-glutathionylation, an increase of which impairs cMyBP-C phosphorylation and increases myofilament calcium sensitivity. In this study, the cysteine modification palmitoylation was investigated, which has not been widely reported for myofilament proteins. Acyl resin assisted capture (Acyl-RAC) was used to purify palmitoylated proteins from cardiac tissue and revealed that actin, myosin and cMyBP-C undergo palmitoylation. Upon investigation of different anatomical regions, cMyBP-C palmitoylation may be highest in the left ventricle and appears reduced when these primary cardiomyocytes are cultured. Furthermore, the palmitoylated form of cMyBP-C may be more resistant to salt extraction from the myofilament lattice. In cardiac pathologies, palmitoylation was reduced in the left ventricle of a rabbit model of heart failure (HF) but increased in ischaemic human HF samples. Site directed mutagenesis revealed C623 and C651, in the C4 and C5 domains respectively, to be candidate palmitoylation sites, which have previously been identified to be modified by S-glutathionylation. Isolated myofilaments treated with palmitoyl CoA, which spontaneously attaches to palmitoylated cysteines, showed significantly increased levels of cMyBP-C palmitoylation and reduced calcium sensitivity of force. Whether this is attributed solely to cMyBP-C palmitoylation remains to be determined, nevertheless this study provides novel evidence that palmitoylation is an important regulatory modification for myofilament function. Aside from palmitoylation, preliminary data suggests cMyBP-C also undergoes SUMOylation. This was investigated using a cMyBP-C-UBC9 fusion construct (WT) co-expressed with eGFP-SUMO1, which shows a SUMOylated band shift, and a catalytically inactive mutant (C93A) which cannot be SUMOylated. Purification of the SUMOylated cMyBP-C-UBC9 fusion for mass spectrometry and in silico analysis identified several candidate SUMOylation sites, however individual mutation did not result in the loss of the SUMOylated band. Reduced phosphorylation of SUMOylated form of cMyBP-C-UBC9 was observed in HEK293 cells and in virally infected neonatal ventricular cardiomyocytes treated with isoprenaline, which also show a blunted lusitropic response to isoprenaline. This may indicate that SUMOylation of cMyBP-C can regulate cardiac contractility, however experimental limitations, including lack of in-situ evidence that cMyBP-C is SUMOylated, limit the conclusions that can be drawn from this study. Given the evidence presented here that cMyBP-C palmitoylation is altered in HF, the palmitoylation of other key cardiac substrates was investigated and were found to be altered in animal models and human HF patients in a similar manner. Animal models of cardiac hypertrophy and HF were generally associated with a loss of palmitoylation, whilst human HF showed increased palmitoylation of substrates including NCX1 and Na+/K+ ATPase. As NCX1 is a reported substrate, expression and palmitoylation of DHHC5 was evaluated in these samples. Cardiac hypertrophy was associated with an increase in DHHC5 expression as early as 3- days post injury, however HF development was associated with unchanged or reduced levels of DHHC5. Previous work suggests DHHC5 overexpression may not directly impact protein palmitoylation or cardiomyocyte function, therefore DHHC5 palmitoylation was evaluated to investigate whether its activity may be changed. Interestingly, DHHC5 palmitoylation followed a similar pattern in disease to NCX1. This may indicate that there are upstream factors such as fatty acid availability that influence the palmitoylation of all substrates together. This study provides an insight into changes of palmitoylation in cardiac disease, although given that changes in singly palmitoylated proteins are more easily detected by Acyl-RAC, further characterisation using additional methods is required

Protein S‐palmitoylation: advances and challenges in studying a therapeutically important lipid modification

The lipid post‐translational modification S‐palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review we discuss the past and recent advances in the experiment tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy

Cyclophilin D palmitoylation and permeability transition: a new twist in the tale of myocardial ischaemia-reperfusion injury

No abstract available

Post-translational regulation of cardiac myosin binding protein-C: a graphical review

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

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740-756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery

Atomic force microscopy—A tool for structural and translational DNA research

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated—providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice

SUMOylation of cardiac myosin binding protein-C reduces its phosphorylation and results in impaired relaxation following treatment with isoprenaline

Systolic and diastolic functions are coordinated in the heart by myofilament proteins that influence force of contraction and calcium sensitivity. Fine control of these processes is afforded by a variety of post-translation modifications that occur on specific proteins at different times during each heartbeat. Cardiac myosin binding protein-C is a sarcomeric accessory protein whose function is to interact transiently with actin, tropomyosin and myosin. Previously many different types of post-translational modification have been shown to influence the action of myosin binding protein-C and we present the first report that the protein can be modified covalently by the small ubiquitin like modifier protein tag. Analysis by mass spectrometry suggests that there are multiple modification sites on myosin binding protein-C for this tag and single point mutations did not serve to abolish the covalent addition of the small ubiquitin like modifier protein. Functionally, our data from both model human embryonic kidney cells and transfected neonatal cardiac myocytes suggests that the modification reduces phosphorylation of the filament protein on serine 282. In cardiac myocytes, the hypo-phosphorylation coincided with a significantly slower relaxation response following isoprenaline induced contraction. We hypothesise that this novel modification of myosin binding protein-C represents a new level of control that acts to alter the relaxation kinetics of cardiac myocytes