Protein Acylation Research Articles

Functions of Coenzyme A and Acyl-CoA in Post-Translational Modification and Human Disease

Coenzyme A (CoA) is synthesized from pantothenate, L-cysteine and adenosine triphosphate (ATP), and plays a vital role in diverse physiological processes. Protein acylation is a common post-translational modification (PTM) that modifies protein structure, function and interactions. It occurs via the transfer of acyl groups from acyl-CoAs to various amino acids by acyltransferase. The characteristics and effects of acylation vary according to the origin, structure, and location of the acyl group. Acetyl-CoA, formyl-CoA, lactoyl-CoA, and malonyl-CoA are typical acyl group donors. The major acyl donor, acyl-CoA, enables modifications that impart distinct biological functions to both histone and non-histone proteins. These modifications are crucial for regulating gene expression, organizing chromatin, managing metabolism, and modulating the immune response. Moreover, CoA and acyl-CoA play significant roles in the development and progression of neurodegenerative diseases, cancer, cardiovascular diseases, and other health conditions. The goal of this review was to systematically describe the types of commonly utilized acyl-CoAs, their functions in protein PTM, and their roles in the progression of human diseases.

MSphere of Influence: The power of polymicrobial partnerships in chronic infection research.

Chelsey VanDrisse works in the field of microbial physiology, studying how acylation of small molecules and proteins affects the development of Pseudomonas biofilms. In this mSphere of Influence article, she reflects on the paper "Community composition shapes microbial-specific phenotypes in a cystic fibrosis polymicrobial model system" by Jean-Pierre et al. This paper prompted her to reassess her approach to studying antibiotic tolerance and her design of experiments that search for disease-relevant mutants and phenotypes in the laboratory.

Differential Abundance of Protein Acylation in Mycobacterium tuberculosis Under Exposure to Nitrosative Stress.

Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM). Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification. A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time. The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health.

Factors Affecting Non-Enzymatic Protein Acylation by trans-3-Methylglutaconyl Coenzyme A.

The leucine catabolism pathway intermediate, trans-3-methylglutaconyl (3MGC) CoA, is considered to be the precursor of 3MGC acid, a urinary organic acid associated with specific inborn errors of metabolism (IEM). trans-3MGC CoA is an unstable molecule that can undergo a sequence of non-enzymatic chemical reactions that lead to either 3MGC acid or protein 3MGCylation. Herein, the susceptibility of trans-3MGC CoA to protein 3MGCylation was investigated. trans-3MGC CoA was generated through the activity of recombinant 3-methylcrotonyl CoA carboxylase (3MCCCase). Following enzyme incubations, reaction mixtures were spin-filtered to remove 3MCCCase. The recovered filtrates, containing trans-3MGC CoA, were then incubated in the presence of bovine serum albumin (BSA). Following this, sample aliquots were subjected to α-3MGC IgG immunoblot analysis to probe for 3MGCylated BSA. Experiments revealed a positive correlation between trans-3MGC CoA incubation temperature and 3MGCylated BSA immunoblot signal intensity. A similar correlation was observed between incubation time and 3MGCylated BSA immunoblot signal intensity. When trans-3MGC CoA hydratase (AUH) was included in incubations containing trans-3MGC CoA and BSA, 3MGCylated BSA immunoblot signal intensity decreased. Evidence that protein 3MGCylation occurs in vivo was obtained in studies with liver-specific 3-hydroxy-3-methylglutaryl (HMG) CoA lyase knockout mice. Therefore, trans-3MGC CoA is a reactive, potentially toxic metabolite, and under normal physiological conditions, lowering trans-3MGC CoA levels via AUH-mediated hydration to HMG CoA protects against aberrant non-enzymatic chemical reactions that lead to protein 3MGCylation and 3MGC acid production.

Insight into the Stabilization Mechanism of Succinylation Modification on Black Bean Protein Gels: Molecular Conformation, Microstructure, and Gel Properties.

The objective of this work was to investigate the effect of succinylation treatment on the physicochemical properties of black bean proteins (BBPI), and the relationship mechanism between BBPI structure and gel properties was further analyzed. The results demonstrated that the covalent formation of higher-molecular-weight complexes with BBPI could be achieved by succinic anhydride (SA). With the addition of SA at 10% (v/v), the acylation of proteins amounted to 92.53 ± 1.10%, at which point there was a minimized particle size of the system (300.90 ± 9.57 nm). Meanwhile, the protein structure was stretched with an irregular curl content of 34.30% and the greatest processable flexibility (0.381 ± 0.004). The dense three-dimensional mesh structure of the hydrogel as revealed by scanning electron microscopy was the fundamental prerequisite for the ability to resist external extrusion. The thermally induced hydrogels of acylated proteins with 10% (v/v) addition of SA showed excellent gel elastic behavior (1.44 ± 0.002 nm) and support capacity. Correlation analysis showed that the hydrogel strength and stability of hydrogels were closely related to the changes in protein conformation. This study provides theoretical guidance for the discovery of flexible proteins and their application in hydrogels.

Role of novel protein acylation modifications in immunity and its related diseases.

The cross-regulation of immunity and metabolism is currently a research hotspot in life sciences and immunology. Metabolic immunology plays an important role in cutting-edge fields such as metabolic regulatory mechanisms in immune cell development and function, and metabolic targets and immune-related disease pathways. Protein post-translational modification (PTM) is a key epigenetic mechanism that regulates various biological processes and highlights metabolite functions. Currently, more than 400 PTM types have been identified to affect the functions of several proteins. Among these, metabolic PTMs, particularly various newly identified histone or non-histone acylation modifications, can effectively regulate various functions, processes and diseases of the immune system, as well as immune-related diseases. Thus, drugs aimed at targeted acylation modification can have substantial therapeutic potential in regulating immunity, indicating a new direction for further clinical translational research. This review summarises the characteristics and functions of seven novel lysine acylation modifications, including succinylation, S-palmitoylation, lactylation, crotonylation, 2-hydroxyisobutyrylation, β-hydroxybutyrylation and malonylation, and their association with immunity, thereby providing valuable references for the diagnosis and treatment of immune disorders associated with new acylation modifications.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

Characterization of diverse lysine acylations in Bacillus thuringiensis: Substrate profiling and functional exploration.

Lysine acylation has been extensively investigated due to its regulatory role in a diverse range of biological functions across prokaryotic and eukaryotic species. In-depth acylomic profiles have the potential to enhance comprehension of the biological implications of organisms. However, the extent of research on global acylation profiles in microorganisms is limited. Here, four lysine acylomes were conducted in Bacillus thuringiensis by using the LC-MS/MS based proteomics combined with antibody-enrichment strategies, and a total of 3438 acetylated sites, 5797 propionylated sites, 1705 succinylated sites, and 925 malonylated sites were identified. The motif analysis of these modified proteins revealed a high conservation of glutamate in acetylation and propionylation, whereas such conservation was not observed in succinylation and malonylation modifications. Besides, conservation analysis showed that homologous acylated proteins in Bacillus subtilis and Escherichia coli were connected with ribosome and aminoacyl-tRNA biosynthesis. Further biological experiments showed that lysine acylation lowered the RNA binding ability of CodY and impaired the in vivo protein activity of MetK. In conclusion, our study expanded the current understanding of the global acylation in Bacillus, and the comparative analysis demonstrated that shared acylation proteins could play important roles in regulating both metabolism and RNA transcription progression.

Amino acid and protein specificity of protein fatty acylation in Caenorhabditis elegans

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S-acyl moieties differ from N- and O-fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S-acylated proteins and 510 hydroxylamine-resistant N- or O-acylated proteins. Subsets of S-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

Highly selective chemical modification of poly-His tagged peptides and proteins.

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.

Metabolic transitions regulate global protein fatty acylation

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration

ABSTRACT The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.

Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

Multi-Method Quantification of Acetyl-Coenzyme A and Further Acyl-Coenzyme A Species in Normal and Ischemic Rat Liver.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.

Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation.

3-methylglutaconyl (3MGC) CoA hydratase (AUH) is the leucine catabolism pathway enzyme that catalyzes the hydration of trans-3MGC CoA to 3-hydroxy, 3-methylglutaryl (HMG) CoA. In several inborn errors of metabolism (IEM), however, metabolic dysfunction can drive this reaction in the opposite direction (the dehydration of HMG CoA). The recent discovery that trans-3MGC CoA is inherently unstable and prone to a series of non-enzymatic chemical reactions provides an explanation for 3MGC aciduria observed in these IEMs. Under physiological conditions, trans-3MGC CoA can isomerize to cis-3MGC CoA, which is structurally poised to undergo intramolecular cyclization with the loss of CoA, generating cis-3MGC anhydride. The anhydride is reactive and has two potential fates; (a) hydrolysis to yield cis-3MGC acid or (b) a reaction with lysine side-chain amino groups to 3MGCylate substrate proteins. An antibody elicited against a 3MGC hapten was employed to investigate protein acylation in incubations containing recombinant AUH, HMG CoA, and bovine serum albumin (BSA). The data obtained show that, as AUH dehydrates HMG CoA to trans-3MGC CoA, BSA is acylated. Moreover, α-3MGC IgG immunoblot signal intensity correlates with AUH concentration, HMG CoA substrate concentration, and incubation time. Thus, protein 3MGCylation may contribute to the phenotypic features associated with IEMs that manifest 3MGC aciduria.

High-Intensity Interval Training Induces Protein Lactylation in Different Tissues of Mice with Specificity and Time Dependence

Protein lysine lactylation (Kla) is a novel protein acylation reported in recent years, which plays an important role in the development of several diseases with pathologically elevated lactate levels, such as tumors. The concentration of lactate as a donor is directly related to the Kla level. High-intensity interval training (HIIT) is a workout pattern that has positive effects in many metabolic diseases, but the mechanisms by which HIIT promotes health are not yet clear. Lactate is the main metabolite of HIIT, and it is unknown as to whether high lactate during HIIT can induce changes in Kla levels, as well as whether Kla levels differ in different tissues and how time-dependent Kla levels are. In this study, we observed the specificity and time-dependent effects of a single HIIT on the regulation of Kla in mouse tissues. In addition, we aimed to select tissues with high Kla specificity and obvious time dependence for lactylation quantitative omics and analyze the possible biological targets of HIIT-induced Kla regulation. A single HIIT induces Kla in tissues with high lactate uptake and metabolism, such as iWAT, BAT, soleus muscle and liver proteins, and Kla levels peak at 24 h after HIIT and return to steady state at 72 h. Kla proteins in iWAT may affect pathways related to glycolipid metabolism and are highly associated with de novo synthesis. It is speculated that the changes in energy expenditure, lipolytic effects and metabolic characteristics during the recovery period after HIIT may be related to the regulation of Kla in iWAT.

Endogenous Cell-Surface Receptor Modification by Metal Chelation-Assisted Pyridinium Oxime Catalyst.

Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of substrates. Here, we report a new catalytic protein acylation strategy promoted by the His-tag/NiNTA interaction. The recognition-assisted activation mechanism allows efficient protein labeling even with >10-fold lower substrate concentrations than conventional reactions, thereby enabling highly selective and efficient cell-surface receptor modification in live cells.

Bioinformatics analysis and experimental validation of cuproptosis-related lncRNA LINC02154 in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is common in urinary system tumors. Cuproptosis is a non-apoptotic cell death pathway. Copper binds to fatty acylated mitochondrial proteins and activates various forms of cell death. LncRNA LINC02154 is significantly highly expressed in cells and tissues of many types of tumors, and the risk signature of LINC02154 in some tumors has been validated for effectiveness.MethodsWe constructed a risk prognostic signature by obtaining differentially expressed long noncoding RNAs (lncRNAs) associated with ccRCC outcomes and cuproptosis from The Cancer Genome Atlas (TCGA). We used TCGA to construct training and testing sets to analyze the risk signature and the impact of LINC02154, and we performed relevant survival analyses. Tumor mutational burdens were analyzed in different LINC02154 expression groups and risk score groups. We next analyzed the immune microenvironment of LINC20154. We performed LINC20154-related drug sensitivity analyses. We also investigated the cellular function of LINC02154 in the ACHN cell line and performed CCK-8 assay, EdU, wound-healing assay, and Transwell assay. The essential genes FDX1 and DLST of cuproptosis were detected by western blot.ResultsWe demonstrated that LINC02154’s impact on outcomes was statistically significant. We also demonstrated the association of different ages, genders, stages, and grades with LINC02154 and risk models. The results showed a significant difference in tumor mutation burden between the groups, which was closely related to clinical prognosis. We found differences in immune cells among groups with different levels of LINC02154 expression and significant differences in immune function, immunotherapeutic positive markers, and critical steps of the immune cycle. The sensitivity analysis showed that differential expression of LINC02154 discriminated between sensitivity to axitinib, doxorubicin, gemcitabine, pazopanib, sorafenib, sunitinib, and temsirolimus. This difference was also present in the high-risk group and low-risk group. We demonstrated that the proliferation and migration of t ACHN cells in the LINC02154 knockdown group were inhibited. The western blot results showed that the knockdown of LINC02154 significantly affected the expression of FDX1 and DLST, critical genes of cuproptosis.ConclusionFinally, we demonstrated that LINC02154 and our constructed risk signature could predict outcomes and have potential clinical value.

Acylation of the rat brain proteins is affected by the inhibition of pyruvate dehydrogenase <i>in vivo</i>

Organism adaptation to metabolic challenges requires coupling of metabolism to gene expression. In this regard, acylations of histones and metabolic proteins acquire significant interest. We hypothesize that adaptive response to inhibition of a key metabolic process, catalyzed by the acetyl-CoA-generating pyruvate dehydrogenase (PDH) complex, is mediated by changes in the protein acylations. The hypothesis is tested by intranasal administration to animals of PDH-specific inhibitors acetyl(methyl)phosphinate (AcMeP) or acetylphosphonate methyl ester (AcPMe), followed by the assessment of physiological parameters, brain protein acylation, and expression/phosphorylation of PDHA subunit. At the same dose, AcMeP, but not AcPMe, decreases acetylation and increases succinylation of the brain proteins with apparent molecular masses of 15-20 kDa. Regarding the proteins of 30-50 kDa, a strong inhibitor AcMeP affects acetylation only, while a less efficient AcPMe mostly increases succinylation. The unchanged succinylation of the 30-50 kDa proteins after the administration of AcMeP coincides with the upregulation of desuccinylase SIRT5. No significant differences between the levels of brain PDHA expression, PDHA phosphorylation, parameters of behavior or ECG are observed in the studied animal groups. The data indicate that the short-term inhibition of brain PDH affects acetylation and/or succinylation of the brain proteins, that depends on the inhibitor potency, protein molecular mass, and acylation type. The homeostatic nature of these changes is implied by the stability of physiological parameters after the PDH inhibition.