Amelioration of nonalcoholic fatty liver disease by inhibiting the deubiquitylating enzyme RPN11.

Nonalcoholic fatty liver disease (NAFLD), including its more severe manifestation nonalcoholic steatohepatitis (NASH), is a global public health challenge. Here, we explore the role of deubiquitinating enzyme RPN11 in NAFLD and NASH. Hepatocyte-specific RPN11 knockout mice are protected from diet-induced liver steatosis, insulin resistance, and steatohepatitis. Mechanistically, RPN11 deubiquitinates and stabilizes METTL3 to enhance the m6A modification and expression of acyl-coenzyme A (CoA) synthetase short-chain family member 3 (ACSS3), which generates propionyl-CoA to upregulate lipid metabolism genes via histone propionylation. The RPN11-METTL3-ACSS3-histone propionylation pathway is activated in the livers of patients with NAFLD. Pharmacological inhibition of RPN11 by Capzimin ameliorated NAFLD, NASH, and related metabolic disorders in mice and reduced lipid contents in human hepatocytes cultured in 2D and 3D. These results demonstrate that RPN11 is a novel regulator of NAFLD/NASH and that suppressing RPN11 has therapeutic potential for the treatment.

A MOZ-TIF2 leukemia mouse model displays KAT6-dependent H3K23 propionylation and overexpression of a set of active developmental genes.

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

Improved Mass Spectrometry-Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles.

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry-based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.

Programmable Acetylation Modification of Bacterial Proteins by a Cas12a-Guided Acetyltransferase.

Protein lysine acetylation (PLA) is a crucial post-translational modification in organisms that regulates a variety of metabolic and physiological activities. Many advances have been made in PLA-related research; however, the quick and accurate identification of causal relationships between specific protein acetylation events and phenotypic outcomes at the proteome level remains challenging due to the lack of efficient targeted modification techniques. In this study, based on the characteristics of transcription-translation coupling in bacteria, we designed and constructed an in situ targeted protein acetylation (TPA) system integrating the dCas12a protein, guiding element crRNA, and bacterial acetylase At2. Rapid identification of multiple independent protein acetylation and cell phenotypic analyses in Gram-negative Escherichia coli and Gram-positive Clostridium ljungdahlii demonstrated that TPA is a specific and efficient targeting tool for protein modification studies and engineering.

Analysis of Lysine Acetylation and Acetylation-like Acylation In Vitro and In Vivo.

Protein lysine acetylation refers to the covalent transfer of an acetyl moiety from acetyl coenzyme A to the epsilon-amino group of a lysine residue and is critical for regulating protein functions in almost all living cells or organisms. Studies in the past decade have demonstrated the unexpected finding that acetylation-like acylation, such as succinylation, propionylation, butyrylation, crotonylation, and lactylation, is also present in histones and many non-histone proteins. Acetylation and acetylation-like acylation serve as reversible on/off switches for regulating protein function while interplaying with other post-translational modifications (such as phosphorylation and methylation) in a codified manner. Lysine acetylation and acetylation-like acylation are important for regulating different cellular and developmental processes in normal and pathological states. Thus, the detection of such modifications is important for related basic research and molecular diagnostics. Traditionally, lysine acetylation is detected by autoradiography, but recent decades have seen great improvement in the quality of site-specific antibodies against acetylation (or acetylation-like acylation), thereby providing competitive alternatives to the use of radioactive acetate and acetyl-coenzyme A for in vivo and in vitro labeling, respectively. This article describes protocols for the detection of lysine acetylation and acetylation-like acylation with site-specific antibodies to complement extant autoradiography-based methods (Pelletier et al., 2017). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acylation assays in vitro Basic Protocol 2: Determination of in vivo acylation.

Propionate reduces the viability of Salmonella enterica Serovar Typhi in macrophages by propionylation of PhoP K102.

Propionate, a major constituent of short chain fatty acids, has recently been reported to be involved in both prokaryotic and eukaryotic lysine propionylation (Kpr). However, the propionylation characteristics of the enteric pathogen Salmonella enterica serovar Typhi (S. Typhi) following invasion of the human gut under the influence of propionate, whether virulence is affected, and the underlying mechanisms are not yet known. In the present study, we report that propionate significantly reduces the viability of S. Typhi in macrophages through intra-macrophage survival assays. We also demonstrate that the concentration of propionate and the propionate metabolic intermediate propionyl coenzyme A can affect the level of modification of PhoP by propionylation, which is tightly linked to intracellular survival. By expressing and purifying PhoP protein in vitro and performing EMSA and protein phosphorylation analyses, We provide evidence that K102 of PhoP is modified by Kpr propionate, which regulates S. Typhi viability in macrophages by decreasing the phosphorylation and DNA-binding ability of PhoP. In conclusion, our study reveals a potential molecular mechanism by which propionate reduces the viability of S. Typhi in macrophages via Kpr.

Global landscape of lysine acylomes in Bacillus subtilis.

Lysine acetylation is a common posttranslational modification that regulates numerous biochemical functions in both eukaryotic and prokaryotic species. In addition, several new non-acetyl acylations are structurally different from lysine acetylation and participate in diverse physiological functions. Here, a comprehensive analysis of several lysine acylomes was performed by combining the high-affinity antibody enrichment with high-resolution LC-MS/MS. In total, we identified 2536 lysine acetylated sites, 4723 propionylated sites, 2150 succinylated sites and 3001 malonylated sites in Bacillus subtilis, respectively. These acylated proteins account for 35.8% of total protein in this bacterium. The four lysine acylomes showed a motif preference for glutamate surrounding the modified lysine residues, and a functional preference for several metabolic pathways, such as carbon metabolism, fatty acid metabolism, and ribosome. In addition, more protein-protein interaction clusters were identified in the propionylated substrates than other three lysine acylomes. In summary, our study presents a global landscape of acylation in the Gram-positive model organism Bacillus and their potential functions in metabolism and physiology.

Quantitative Analysis of Posttranslational Modifications of Plant Histones.

Reshaping of the chromatin landscape under oxidative stress is of paramount importance for mounting an effective stress response. Unbiased systemic identification and quantification of histone marks is crucial for understanding the epigenetic component of plant responses to adverse environmental conditions. We describe a detailed method for isolation of plant histones and subsequent bottom-up proteomics approach for characterization of acetylation and methylation status. By performing label-free quantitative mass spectrometry analysis, relative abundances of histone marks can be statistically compared between experimental conditions.

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation.

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Manipulation of the internal structure of starch by propionyl treatment and its diverse influence on digestion and in vitro fermentation characteristics.

High amylose maize starch (HAMS) and waxy maize starch (WMS) were modified by propionylation and their corresponding physicochemical characteristics, digestion and fermentation properties were studied. The results indicated that two new peaks related to methylene (2.20 ppm) and methyl (0.97 ppm) in the NMR spectrum were formed, indicating the occurrence of propionylation, and this was further confirmed by the formation of a characteristic absorption at 1747 cm-1 in the FTIR spectrum. The propionylation led the modified starch having a lower electron density contrast between the crystalline and amorphous flakes, resulting in the formation of a more compact structure following the increased degrees of substitution (DS). The propionylated starch also had a higher thermal stability and hydrophobicity. These structural changes increased the content of resistant starch (RS) and reduced the predicted glycemic index. More importantly, the gut microbiota fermentation properties indicated that the propionylation of the starch can not only increase the yield of propionate, but also increase the concentration of total short-chain fatty acids (SCFAs). This study highlights a new approach to significantly enhance the RS content in starch, together with an increased SCFA generation capacity.

Effect of the Propionylation Method on the Deformability under Thermal Pressure of Block-Shaped Wood.

Converting wood waste into thermoplastic materials is an attractive means of increasing its utilization because complex three-dimensional molded products can easily be obtained by press molding wood with thermoplasticity. Chemical modification, especially esterification, is a promising method for imparting thermoplasticity to wood. In this study, we produced multiple propionylated wood specimens using several propionylation methods and elucidated the factors affecting the deformability of the wood. Regardless of the method, all of the propionylated wood samples showed deformability in the tangential direction. However, in the longitudinal direction, not only the degree of propionylation but also the propionylation method had a significant influence on the deformability. The flow in the tangential direction occurred under a relatively low pressure, whereas the flow in the longitudinal direction occurred under higher pressure. The chemical composition and motility of each sample were evaluated using solid-state NMR measurements. With some propionylation methods, decomposition of the cellulose main chain occurred during the reaction, which had a dominant effect on the deformability of the wood in the longitudinal direction. These results indicate that the deformability of wood can be controlled by the appropriate selection of a propionylation method and its treatment conditions.

Detection of single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of Phi29 DNA packaging motor.

Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM.

Propionate hampers differentiation and modifies histone propionylation and acetylation in skeletal muscle cells.

Protein acylation via metabolic acyl-CoA intermediates provides a link between cellular metabolism and protein functionality. A process in which acetyl-CoA and acetylation are fine-tuned is during myogenic differentiation. However, the roles of other protein acylations remain unknown. Protein propionylation could be functionally relevant because propionyl-CoA can be derived from the catabolism of amino acids and fatty acids and was shown to decrease during muscle differentiation. We aimed to explore the potential role of protein propionylation in muscle differentiation, by mimicking a pathophysiological situation with high extracellular propionate which increases propionyl-CoA and protein propionylation, rendering it a model to study increased protein propionylation. Exposure to extracellular propionate, but not acetate, impaired myogenic differentiation in C2C12 cells and propionate exposure impaired myogenic differentiation in primary human muscle cells. Impaired differentiation was accompanied by an increase in histone propionylation as well as histone acetylation. Furthermore, chromatin immunoprecipitation showed increased histone propionylation at specific regulatory myogenic differentiation sites of the Myod gene. Intramuscular propionylcarnitine levels are higher in old compared to young males and females, possibly indicating increased propionyl-CoA levels with age. The findings suggest a role for propionylation and propionyl-CoA in regulation of muscle cell differentiation and ageing, possibly via alterations in histone acylation.

Profile of protein lysine propionylation in Aeromonas hydrophila and its role in enzymatic regulation.

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.

A Transfer Learning-Based Approach for Lysine Propionylation Prediction.

Lysine propionylation is a newly discovered posttranslational modification (PTM) and plays a key role in the cellular process. Although proteomics techniques was capable of detecting propionylation, large-scale detection was still challenging. To bridge this gap, we presented a transfer learning-based method for computationally predicting propionylation sites. The recurrent neural network-based deep learning model was trained firstly by the malonylation and then fine-tuned by the propionylation. The trained model served as feature extractor where protein sequences as input were translated into numerical vectors. The support vector machine was used as the final classifier. The proposed method reached a matthews correlation coefficient (MCC) of 0.6615 on the 10-fold crossvalidation and 0.3174 on the independent test, outperforming state-of-the-art methods. The enrichment analysis indicated that the propionylation was associated with these GO terms (GO:0016620, GO:0051287, GO:0003735, GO:0006096, and GO:0005737) and with metabolism. We developed a user-friendly online tool for predicting propoinylation sites which is available at http://47.113.117.61/.

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.

Rice Histone Propionylation and Generation of Chemically Derivatized Synthetic H3 and H4 Peptides for Identification of Acetylation Sites and Quantification.

Histone proteins are crucial in the study of chromatin dynamics owing to their wide-ranging implications in the regulation of gene expression. Modifications of histones are integral to these regulatory processes in concert with associated proteins, such as transcription factors and coactivators. One of the biochemical techniques available to enhance analysis of histone proteins is chemical derivatization using propionic anhydride. In this protocol, we describe the use of propionylation to efficiently derivatize acid-extracted histones from rice. We also synthesize H3 and H4 tryptic peptides, thus mimicking the nature of derivatized extracted peptides to aid in identification and quantification using targeted-mass spectrometry. Here we make available the masses of the precursor ions and the retention times (RT) of each synthesized peptide. These provide useful information to facilitate histone data analysis. Lastly, we note that we will distribute these synthetic peptides in nanomolar (nM) concentrations to those who wish to utilize them for assays and further experimental studies.

Lysine malonylation and propionylation are prevalent in human lens proteins.

Acylated lysine residues represent major chemical modifications in proteins. We investigated the malonylation and propionylation of lysine residues (MalK, PropK) in the proteins of aging human lenses. Western blot results showed that the two modifications are present in human lens proteins. Liquid chromatography-mass spectrometry (LC-MS/MS) results showed 4-18 and 4-32 pmol/mg protein of MalK and PropK, respectively, in human lens proteins with no apparent changes related to aging. Mass spectrometry results revealed that MalK- and PropK-modified lysine residues are present in all major crystallins, other cytosolic proteins, and membrane and cytoskeletal proteins of the lens. Several mitochondrial and cytosolic proteins in cultured human lens epithelial cells showed MalK and PropK modifications. Sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) were present in human lens epithelial and fiber cells. Moreover, lens epithelial cell lysate deacylated propionylated and malonylated lysozyme. The absence of SIRT3 and SIRT5 led to higher PropK and MalK levels in mouse lenses. Together, these data suggest that MalK and PropK are widespread modifications in lens and SIRT3 and SIRT5 could regulate their levels in lens epithelial cells.

Proteome-Wide Identification of Lysine Propionylation in the Conidial and Mycelial Stages of Trichophyton rubrum.

Posttranslational modifications (PTMs) exist in a wide variety of organisms and play key roles in regulating various essential biological processes. Lysine propionylation is a newly discovered PTM that has rarely been identified in fungi. Trichophyton rubrum (T. rubrum) is one of the most common fungal pathogens in the world and has been studied as an important model organism of anthropic pathogenic filamentous fungi. In this study, we performed a proteome-wide propionylation analysis in the conidial and mycelial stages of T. rubrum. A total of 157 propionylated sites on 115 proteins were identified, and the high confidence of propionylation identification was validated by parallel reaction monitoring (PRM) assay. The results show that the propionylated proteins were mostly involved in various metabolic pathways. Histones and 15 pathogenicity-related proteins were also targets for propionylation modification, suggesting their roles in epigenetic regulation and pathogenicity. A comparison of the conidial and mycelial stages revealed that most propionylated proteins and sites were growth-stage specific and independent of protein abundance. Based on the function classifications, the propionylated proteins had a similar distribution in both stages; however, some differences were also identified. Furthermore, our results show that the concentration of propionyl-CoA had a significant influence on the propionylation level. In addition to the acetylation, succinylation and propionylation identified in T. rubrum, 26 other PTMs were also found to exist in this fungus. Overall, our study provides the first global propionylation profile of a pathogenic fungus. These results would be a foundation for further research on the regulation mechanism of propionylation in T. rubrum, which will enhance our understanding of the physiological features of T. rubrum and provide some clues for the exploration of improved therapies to treat this medically important fungus.

Lysine Propionylation is a Widespread Post-Translational Modification Involved in Regulation of Photosynthesis and Metabolism in Cyanobacteria.

Lysine propionylation is a reversible and widely distributed post-translational modification that is known to play a regulatory role in both eukaryotes and prokaryotes. However, the extent and function of lysine propionylation in photosynthetic organisms remains unclear. Cyanobacteria are the most ancient group of Gram-negative bacteria capable of oxygenic photosynthesis, and are of great importance to global carbon and nitrogen cycles. Here, we carried out a systematic study of lysine propionylaiton in cyanobacteria where we used Synechocystis sp. PCC 6803 (Synechocystis) as a model. Combining high-affinity anti-propionyllysine pan antibodies with high-accuracy mass spectrometry (MS) analysis, we identified 111 unique lysine propionylation sites on 69 proteins in Synechocystis. Further bioinformatic analysis showed that a large fraction of the propionylated proteins were involved in photosynthesis and metabolism. The functional significance of lysine propionylation on the enzymatic activity of fructose-1,6-bisphosphatase (FbpI) was studied by site-directed mutagenesis and biochemical studies. Further functional studies revealed that the propionylation level of subunit II of photosystem I (PsaD) was obviously increased after high light (HL) treatment, suggesting that propionylation may be involved in high light adaption in Synechocystis. Thus, our findings provide novel insights into the range of functions regulated by propionylation and reveal that reversible propionylation is a functional modification with the potential to regulate photosynthesis and carbon metabolism in Synechocystis, as well as in other photosynthetic organisms.