Proteomic insights into lysine acetylation and the implications for medical research

KEYWORDS:

• HATs
• HDACs
• lysine acetylation
• medical research
• proteomics

1. Introduction

Lysine acetylation is a posttranslational modification (PTM) involved in almost every cellular process in eukaryotic cells, including central metabolism, remodeling of chromatin, transcriptional activation, protein stabilization, and subcellular localization, among others [1].

A lysine residue becomes acetylated by the action of the histone/lysine acetyltransferase enzymes (HATs/KATs) and is removed by histone/lysine deacetylases (HDACs/KDACs). However, it has been reported that in the mitochondrial matrix, can occur nonenzymatically [2]. The enzymatic acetylation process occurs by the transfer of an acetyl group from AcCoA to the epsilon amino group in the lysine residue by HATs. In contrast, the deacetylation process consists of the removal of the acetyl group from the lysine residue, which can be achieved by HDACs which use Zn²+ as cofactor, or by sirtuins, a specific of NAD+ dependent form of HDACs [3].

High-resolution mass spectrometry-based proteomics has been used to significantly increase our knowledge of acetylation and its potential inferences in numerous diseases. An amalgamation of diverse proteomic strategies will provide a wide-ranging characterization of acetylation in several experimental conditions [4]. For the identification and quantification of acetylated peptides, methods based on the immunoprecipitation of acetylated lysine-containing peptides to enrich acetylated peptides should be used. Quantitative proteomic analysis is essential to find out the relative abundance of protein expression providing information regarding protein expression in several diseases. Groups of proteins that regulate the acetylation of lysine deserve special attention because numerous components of these cluster are deregulated in several diseases, as we will describe below. In addition, the acetylation stoichiometric analysis has been enriching our knowledge of the amount of acetylation for each residue in different pathologies. In addition, specific inhibitors of certain regulators of acetylation can be used to identify their possible substrates and to evaluate their global effect, increasing our knowledge of how this PTM is involved in cellular process. Consequently, proteomics methodologies have been carried out to investigate proteins and thousands of acetylation sites with implications in several biological process in health and in disease [4].

2. Lysine acetylation and its role in human disorders

Different KATs and HDACs have been implicated in a wide range of cellular processes. A disparity of this regulation could lead to the development of diverse diseases such as cancer, diabetes, viral infections, neurodegenerative diseases like Alzheimer´s (AD), Parkinson’s, and disorders related with developmental abnormalities, just to mention some relevant pathologies.

In AD, acetylation of Tau, impairs Tau-mediated stabilization, promoting pathological Tau aggregation, consequently Tau acetylation at Lys 174 is actually identified as an early marker of AD [5]. Additionally, tubulin acetylation is a hallmark for long-lived microtubules, and HDAC6 is responsible for the removal of this modification. When HDAC6 is silenced in mice, α-tubulin is hyperacetylated in almost every tissue. These mutants display impaired immune responses, hyperactivity, decreased anxiety and lower depression tendency [6].

Parkinson´s disease is another neurodegenerative condition that affects the motor system of the central nervous system. Mutations of the LRRK2 (leucine-rich repeat kinase 2) gene are the most common genetic source of this disease [7]. Imperfect microtubule-based axonal transport is one possible cause. Associated with this, LRRK2 interacts directly with β-tubulin and inhibits α-tubulin acetylation [8], which indicates that in normal cellular contexts, this kinase acts as a negative regulator of microtubule acetylation. Contrasting wild-type LRRK2, two mutants carrying Parkinson’s disease-associated mutations form filamentous subcellular structures [4]. Expression of ATAT1 or inhibition of HDAC6 prevents formation of such structures. Also, tubulin acetylation has been associated with other neurological disorders like Charcot-Marie-Tooth disease, and Huntington’s disease, where HDAC6 inhibits intracellular vesicular transport [9].

Acetylation and deacetylation also take part in different viral mechanisms that facilitate the infection of host cells. Human immunodeficiency virus (HIV) infection stabilizes microtubules and promotes tubulin acetylation in host cells; thereby overexpression of HDAC6 inhibits HIV infection. On the other hand, Influenza A virus infection is inhibited by the action of HDAC6, which downregulates the trafficking of viral components to the host cell plasma membrane through acetylated microtubules [10].

In cancer, lysine acetylation plays an important role in regulating protein function and localization. Deregulation in KATs, HDACs, and lysine acetylation readers, had been reported, for example, GC5N with demonstrated participation in Leukemia cells and neuroblastoma cells, PCAF in T-cell lymphoma, lung adenocarcinoma, prostate cancer, among others), CBP/p300 (prostate cancer cells, glioma, melanoma cells, renal cell carcinoma), Tip60 (breast cancer cells, squamous cell carcinoma, among others), just to mention some examples of KATS activities in cancer development [11]. On the other hand, the deregulation of HDAC6 regulates angiogenesis, cell migration and chemiotaxis, and promotes the expression of genes in prostate cancer; SIRT1 promotes proteasomal degradation of p53 and negatively regulates c-MYC target genes; SIRT3 negatively regulates the production of reactive oxygen species and the stability of HIF1a [3].

In cardiac biology, a role for lysine acetylation was reported in the maintenance of redox balance, cardiac substrate selection and bioenergetic performance. Additionally, in the heart, HDAC inhibitors have been shown to decrease pressure overload-driven interstitial cardiac fibrosis and to reverse pre-established atrial fibrosis and arrhythmic inducibility in Hop transgenic mice [12]. Lysine acetylation was identified as an endogenous regulator of the proteolytic activity in the heart. The inhibition of HDACs was effective in changing the acetylation profile of 20S proteasome complexes and enhancing the proteolytic function of healthy and unhealthy murine and human myocardium [13]. So, it can be said that cardiac proteasomal function could be regulated by HDAC inhibitors.

HDACs (1,2, and 3) has been linked to modulation of the immune response and immunological signaling, since lysine acetylation regulates negatively and positively interferon and TLR signaling by targeting both histone and non-histone proteins, signaling pathways that are linked with responses to pathogen infection, antigen presentation, and chronic inflammation. In addition, we must add its role in the regulation of lipid metabolism and the accumulation of toxic lipid mediators, are involved in a process called inmunometabolic modulation, indicating a central role for lysine acetylation in obesity and associated metabolic diseases, where KATs and HDACs regulate insulin sensitivity by modulating the acetylation of proteins in the insulin signaling cascade, and of other proteins involved in glucose, lipid, and carbon metabolism [14].

Mutations in several HDACs and KATs are also implicated in disorders related with developmental abnormalities; mutations in SMC3 and HDAC8 genes are related with Cornelia de Lange syndrome, and mutations in the ESCO2 gene that codes for the acetyltransferase of SMC3, are associated with Roberts syndrome. Also, Rubinstein–Taybi syndrome is produced by mutations in EP300 and CREBBP [15], in addition, KAT6A mutations are related with developmental delays and intellectual disability, and genitopatellar syndrome and Ohdo syndromes by mutations in KAT6B [16].

3. Acetylation as a therapeutic target

To date, wide-range KATs, class I and II HDAC, sirtuins, and bromodomain inhibitors have been considered and utilized as anti-cancer products. HDAC inhibitors can be classified as members of five classes of compounds: benzamides, cyclic tetrapeptides, hydroxamates, short chain fatty (aliphatic) acids, and sirtuin inhibitors. HDAC inhibitors vorinostat, romidepsin, and belinostat have been approved by the United States Food and Drug Administration for some T-cell lymphoma, and panobinostat for multiple myeloma. The short chain fatty acids, valproic acid (VPA), butyric acid, and phenylbutyric acid, are known to be weak inhibitors of HDAC class I and II. VPA is registered for therapy in bipolar disorders and epilepsy, and with other short chain fatty acids HDAC inhibitors as anticancer drugs, utilized for the treatment of cutaneous T-cell lymphoma. Although, the mechanism of action of all the inhibitors has not been completely determined, HDAC inhibitors modulate immune response, induce cell cycle arrest, apoptosis, differentiation and cell death, and reduce angiogenesis. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies [17].

Currently, clinical trials of HDACs inhibitors have been focused mainly on cancer treatments, but have potential effects against other nonmalignant diseases, such as osteoporosis, spinal muscular atrophy, AD, Parkinson, and Huntington´s diseases, have also been utilized for their antiviral effects as vorinostat, which dislocates HIV-1 latency in patients treated with antiretroviral therapies. These inhibitors were also shown to enhance memory formation and synaptic plasticity in mice [16].

However, in either case the inhibitors have the inconvenience of having a very global mechanism of action and thus affect all the acetylated target proteins and, in consequence; cytoskeleton organization, autophagy, RNA processing and stability, protein folding, protein aggregation, protein degradation and protein-protein interactions in almost every cell in the body. They even affect other PTMs like succinylation, propionylation, ubiquitylation, and phosphorylation [4]. As a consequence, a current goal is the development of new inhibitors that could be combined with other medications and/or radiotherapy to improve their action over those achieved with the inhibitor alone. This could lead to molecules with defined targets, and enhanced therapeutic effects with no or minimal side effects.

4. Concluding remarks

Acetylation is crucial in several cellular metabolic pathways and consequently central to different human disorders. Although there is still much to know about their specific roles in different diseases, no doubt subsequent investigations using several proteomics methods will reveal their fundamental roles in pathological processes, turning the KATs and HDACs and even to the acetylated residues and the stoichiometry of these acetylations into keys for understanding diseases and developing therapeutic and diagnostic targets.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This work was in part supported by grants from Consejo Nacional de Ciencia y Tecnología: 220790 and DGAPA-PAPIIT: IN213216.