Cysteine Residues In Proteins Research Articles

Regulation of Cell Polarity by Posttranslational Protein Palmitoylation.

Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.

Allicin, an Antioxidant and Neuroprotective Agent, Ameliorates Cognitive Impairment.

Allicin (diallylthiosulfinate) is a defense molecule produced by cellular contents of garlic (Allium sativum L.). On tissue damage, the non-proteinogenic amino acid alliin (S-allylcysteine sulfoxide) is converted to allicin in an enzyme-mediated process catalysed by alliinase. Allicin is hydrophobic in nature, can efficiently cross the cellular membranes and behaves as a reactive sulfur species (RSS) inside the cells. It is physiologically active molecule with the ability to oxidise the thiol groups of glutathione and between cysteine residues in proteins. Allicin has shown anticancer, antimicrobial, antioxidant properties and also serves as an efficient therapeutic agent against cardiovascular diseases. In this context, the present review describes allicin as an antioxidant, and neuroprotective molecule that can ameliorate the cognitive abilities in case of neurodegenerative and neuropsychological disorders. As an antioxidant, allicin fights the reactive oxygen species (ROS) by downregulation of NOX (NADPH oxidizing) enzymes, it can directly interact to reduce the cellular levels of different types of ROS produced by a variety of peroxidases. Most of the neuroprotective actions of allicin are mediated via redox-dependent pathways. Allicin inhibits neuroinflammation by suppressing the ROS production, inhibition of TLR4/MyD88/NF-κB, P38 and JNK pathways. As an inhibitor of cholinesterase and (AChE) and butyrylcholinesterase (BuChE) it can be applied to manage the Alzheimer’s disease, helps to maintain the balance of neurotransmitters in case of autism spectrum disorder (ASD) and attention deficit hyperactive syndrome (ADHD). In case of acute traumatic spinal cord injury (SCI) allicin protects neuron damage by regulating inflammation, apoptosis and promoting the expression levels of Nrf2 (nuclear factor erythroid 2-related factor 2). Metal induced neurodegeneration can also be attenuated and cognitive abilities of patients suffering from neurological diseases can be ameliorates by allicin administration.

Pillars and Gaps of S-Nitrosylation-Dependent Epigenetic Regulation in Physiology and Cancer.

Nitric oxide (NO) is a diffusible signaling molecule produced by three isoforms of nitric oxide synthase, which release NO during the metabolism of the amino acid arginine. NO participates in pathophysiological responses of many different tissues, inducing concentration-dependent effect. Indeed, while low NO levels generally have protective effects, higher NO concentrations induce cytotoxic/cytostatic actions. In recent years, evidences have been accumulated unveiling S-nitrosylation as a major NO-dependent post-translational mechanism ruling gene expression. S-nitrosylation is a reversible, highly regulated phenomenon in which NO reacts with one or few specific cysteine residues of target proteins generating S-nitrosothiols. By inducing this chemical modification, NO might exert epigenetic regulation through direct effects on both DNA and histones as well as through indirect actions affecting the functions of transcription factors and transcriptional co-regulators. In this light, S-nitrosylation may also impact on cancer cell gene expression programs. Indeed, it affects different cell pathways and functions ranging from the impairment of DNA damage repair to the modulation of the activity of signal transduction molecules, oncogenes, tumor suppressors, and chromatin remodelers. Nitrosylation is therefore a versatile tool by which NO might control gene expression programs in health and disease.

Hypothiocyanous acid oxidizes collapsin response mediator protein 2 in brain pericytes

AbstractBackgroundPericytes cover brain endothelium to enable maintenance of the blood‐brain barrier (BBB). Decreasing pericyte numbers are reported in Alzheimer’s disease and could lead to BBB dysfunction, triggering vascular inflammation and the recruitment of immune cells. Neutrophils contain high contents of myeloperoxidase, a heme enzyme that produces several oxidising species, including hypothiocyanous acid (HOSCN). HOSCN specifically targets cysteine residues in proteins and low molecular weight thiols, and is more stable than other oxidants in biological systems. A microtubule stabilising protein, collapsin response mediator protein 2 (CRMP2), is oxidised by hydrogen peroxide (H2O2) in some cell types, purportedly through a peroxiredoxin (Prx) relay mechanism. Here, we have tested whether HOSCN can oxidise CRMP2 and lead to disruption of microtubules (MT) in pericytes.MethodMurine pericytes were harvested and cultured, then treated with sub‐lethal concentrations of HOSCN and H2O2. Western blotting was used to observe CRMP2 and Prx oxidation, along with CRMP2 phosphorylation at sites important for MT binding. Fluorescent staining, live‐cell imaging and in vitro scratch assays were used to view cytoskeleton alterations and pericyte migration. Immunoprecipitation and proximity ligation assays were used to investigate physical associations between CRMP2 and Prxs.ResultTreatment of pericytes with HOSCN led to maximal CRMP2 oxidation after 60 min, whereas H2O2 reaches this in 5 min. Oxidation was closely associated with pericyte shrinkage, less growing microtubules and lower pericyte cell migration. CRMP2 oxidation occurred at the same time as Prx2 oxidation, and there was a physical association between these proteins, but it is not possible to conclude a redox relay occurs.ConclusionWe hypothesized that the longer‐lived oxidant HOSCN could oxidize CRMP2 and impair pericyte function at sub‐lethal doses. Indeed, HOSCN led to less dynamic MTs and subsequent lower migration of pericytes, likely lowering their ability to cover empty area on the inflamed vasculature to protect endothelial function. This effect is reversible upon HOSCN removal, nevertheless HOSCN could be a cause of BBB disruption during neuroinflammation. The mechanisms of CRMP2 oxidation are currently being investigated. Strategies that protect this MT regulatory protein from oxidation could have therapeutic benefit in neurological disease.

Drug-drug interactions induced by Linderane based on mechanism-based inactivation of CYP2C9 and the molecular mechanisms

Linderane (LDR) is a main furan-containing sesquiterpenoid of the common herbal medicine Lindera aggregata (Sims) Kosterm. Our early study indicated that LDR led to mechanism-based inactivation (MBI) of CYP2C9 in vitro, implying possible drug-drug interactions (DDIs) in clinic. In the present study, influence of LDR on the pharmacokinetics of the corresponding hydroxylated metabolites of CYP2C9 substrates in rats was investigated. Pharmacokinetic studies revealed that pretreatment with LDR at 20 mg/kg for 15 days inhibited the metabolism of both tolbutamide and warfarin catalyzed by CYP2C9. As for 4-hydroxytolbutamide, the Cmax was decreased, the t1/2z was prolonged, and the Vz/F was increased, all with significant difference. As for 7-hydroxywarfarin, the AUC0-t/AUC0-∞ and CLz/F were significantly decreased and increased, respectively. Furthermore, the underlying molecular mechanisms based on MBI of CYP2C9 by LDR were revealed. Two reactive metabolites of LDR, furanoepoxide and γ-ketoenal intermediates were identified in CYP2C9 recombinant enzyme incubation systems. Correspondingly, covalent modifications of lysine and cysteine residues of CYP2C9 protein were discovered in the CYP2C9 incubation system treated with LDR. The formation of protein adducts exhibited obvious time- and dose-dependence, which is consistent with the trend of enzyme inhibition caused by LDR in vitro. In addition to the apoprotein of CYP2C9, the heme content was significantly reduced after co-incubation with LDR. These data revealed that modification of both apoprotein and heme of CYP2C9 by reactive metabolites of LDR led to MBI of CYP2C9, therefore resulting in the inhibition of biotransformation of CYP2C9 substrates to their corresponding metabolites in vivo.

A sticky situation: regulation and function of protein palmitoylation with a spotlight on the axon and axon initial segment.

In neurons, the axon and axon initial segment (AIS) are critical structures for action potential initiation and propagation. Their formation and function rely on tight compartmentalisation, a process where specific proteins are trafficked to and retained at distinct subcellular locations. One mechanism which regulates protein trafficking and association with lipid membranes is the modification of protein cysteine residues with the 16-carbon palmitic acid, known as S-acylation or palmitoylation. Palmitoylation, akin to phosphorylation, is reversible, with palmitate cycling being mediated by substrate-specific enzymes. Palmitoylation is well-known to be highly prevalent among neuronal proteins and is well studied in the context of the synapse. Comparatively, how palmitoylation regulates trafficking and clustering of axonal and AIS proteins remains less understood. This review provides an overview of the current understanding of the biochemical regulation of palmitoylation, its involvement in various neurological diseases, and the most up-to-date perspective on axonal palmitoylation. Through a palmitoylation analysis of the AIS proteome, we also report that an overwhelming proportion of AIS proteins are likely palmitoylated. Overall, our review and analysis confirm a central role for palmitoylation in the formation and function of the axon and AIS and provide a resource for further exploration of palmitoylation-dependent protein targeting to and function at the AIS.

Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways

Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid β-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.

Cysteine trisulfide oxidizes protein thiols and induces electrophilic stress in human cells

The cellular effects of hydrogen sulfide (H2S) signaling may be partially mediated by the formation of alkyl persulfides from thiols, such as glutathione and protein cysteine residues. Persulfides are potent nucleophiles and reductants and therefore potentially an important endogenous antioxidant or protein post-translational modification. To directly study the cellular effects of persulfides, cysteine trisulfide (Cys-S3) has been proposed as an in situ persulfide donor, as it reacts with cellular thiols to generate cysteine persulfide (Cys-S-S−). Numerous pathways sense and respond to electrophilic cellular stressors to inhibit cellular proliferation and induce apoptosis, however the effect of Cys-S3 on the cellular stress response has not been addressed. Here we show that Cys-S3 inhibited cellular metabolism and proliferation and rapidly induced cellular- and ER-stress mechanisms, which were coupled to widespread protein-thiol oxidation. Cys-S3 reacted with Na2S to generate cysteine persulfide, which protected human cell lines from ER-stress. However this method of producing cysteine persulfide contains excess sulfide, which interferes with the direct analysis of persulfide donation. We conclude that cysteine trisulfide is a thiol oxidant that induces cellular stress and decreased proliferation.

Glutaredoxin-1 levels in plasma can predict future events in patients with cardiovascular diseases

Reactive oxygen species that increase during cardiovascular disease (CVD) react with protein cysteine residues to form a glutathione adduct by S-glutathionylation, which is selectively removed by glutaredoxin-1 (Glrx). We previously showed that S-glutathionylation and Glrx play important roles in mouse models of CVD, such as heart failure and peripheral artery disease models. However, there are few clinical studies on Glrx in CVD. Although Glrx is a cytosolic protein expressed in various organs, it is detectable in human plasma. Studies have reported that Glrx in plasma is a potential disease maker, such as CVD and chronic kidney disease and diabetes, however, it remains unclear whether Glrx is related to the prognosis of patients with CVD. The purpose of this study was to elucidate whether Glrx levels in plasma are associated with future events in patients with CVD. Plasma levels of Glrx were measured in 555 patients with CVD who underwent cardiac catheterization using enzyme-linked immunosorbent assay. All patients were followed prospectively for ≤36 months or until occurrence of adverse events, including all-cause death, non-fatal myocardial infarction, and worsening heart failure. During a mean follow-up period of 33 months, 54 adverse events occurred. Kaplan-Meier analysis showed that higher levels of Glrx (>0.622 ng/mL, determined by receiver-operating characteristic curve) resulted in a higher probability for adverse events compared with lower levels of Glrx (≤0.622 ng/mL) (P < 0.01, log-rank test). Multivariate Cox proportional hazards analysis showed that Glrx was a significant predictor of adverse events after adjustment for known risk factors. In conclusion, levels of plasma Glrx >0.662 ng/mL can predict future events in patients with CVD.

Human Cystathionine γ-Lyase Is Inhibited by s-Nitrosation: A New Crosstalk Mechanism between NO and H2S.

The ‘gasotransmitters’ hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

Coenzyme a Biochemistry: From Neurodevelopment to Neurodegeneration.

Coenzyme A (CoA) is an essential cofactor in all living organisms. It is involved in a large number of biochemical processes functioning either as an activator of molecules with carbonyl groups or as a carrier of acyl moieties. Together with its thioester derivatives, it plays a central role in cell metabolism, post-translational modification, and gene expression. Furthermore, recent studies revealed a role for CoA in the redox regulation by the S-thiolation of cysteine residues in cellular proteins. The intracellular concentration and distribution in different cellular compartments of CoA and its derivatives are controlled by several extracellular stimuli such as nutrients, hormones, metabolites, and cellular stresses. Perturbations of the biosynthesis and homeostasis of CoA and/or acyl-CoA are connected with several pathological conditions, including cancer, myopathies, and cardiomyopathies. In the most recent years, defects in genes involved in CoA production and distribution have been found in patients affected by rare forms of neurodegenerative and neurodevelopmental disorders. In this review, we will summarize the most relevant aspects of CoA cellular metabolism, their role in the pathogenesis of selected neurodevelopmental and neurodegenerative disorders, and recent advancements in the search for therapeutic approaches for such diseases.

A novel yeast-based high-throughput method for the identification of protein palmitoylation inhibitors.

Protein S-acylation or palmitoylation is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of proteins through a thioester bond. Palmitoylation and palmitoyltransferases (PATs) have been linked to several types of cancers, diseases of the central nervous system and many infectious diseases where pathogens use the host cell machinery to palmitoylate their effectors. Despite the central importance of palmitoylation in cell physiology and disease, progress in the field has been hampered by the lack of potent-specific inhibitors of palmitoylation in general, and of individual PATs in particular. Herein, we present a yeast-based method for the high-throughput identification of small molecules that inhibit protein palmitoylation. The system is based on a reporter gene that responds to the acylation status of a palmitoylation substrate fused to a transcription factor. The method can be applied to heterologous PATs such as human DHHC20, mouse DHHC21 and also a PAT from the parasite Giardia lamblia. As a proof-of-principle, we screened for molecules that inhibit the palmitoylation of Yck2, a substrate of the yeast PAT Akr1. We tested 3200 compounds and were able to identify a candidate molecule, supporting the validity of our method.

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide‐binding tag site‐specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time‐resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100‐fold enhancement in luminescence quantum yield, affording a highly sensitive turn‐on luminescence probe for time‐resolved FRET assays and enzyme reaction monitoring.

Functional inhibition of redox regulated heme proteins: A novel mechanism towards oxidative stress induced by homocysteine

Homocysteine (Hcy) is a sulfur containing non-protein toxic amino acid synthesized from methionine. Elevated level of Hcy is associated with cardiovascular complications and neurodegeneration. Hcy is believed to induce organ damage and apoptosis via oxidative stress. The pro-oxidant nature of Hcy is considered to originate from the metal-induced oxidation of thiol group-containing molecules forming disulfides (Hcy-Hcy, Hcy-cysteine, Hcy-glutathione, etc) or with free cysteine residues of proteins (a process called protein S-homocysteinylation). Formation of such disulfides indeed results in the generation of reactive oxygen species (ROS) which eventually leads to loss of cellular integrity. In the present manuscript, we performed systematic investigation of the effect of Hcy on iron containing proteins. We discover a novel mechanism of Hcy toxicity wherein Hcy oxidation is linked with the functional loss of the protein with iron as cofactors. Our results indicate that redox regulated heme proteins might be primarily involved in the Hcy toxicity and associated oxidative stress.

Cysteine Proteome Reveals Response to Endogenous Oxidative Stress in Bacillus cereus.

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.

Biological Mechanisms of S-Nitrosothiol Formation and Degradation: How Is Specificity of S-Nitrosylation Achieved?

The modification of protein cysteine residues underlies some of the diverse biological functions of nitric oxide (NO) in physiology and disease. The formation of stable nitrosothiols occurs under biologically relevant conditions and time scales. However, the factors that determine the selective nature of this modification remain poorly understood, making it difficult to predict thiol targets and thus construct informatics networks. In this review, the biological chemistry of NO will be considered within the context of nitrosothiol formation and degradation whilst considering how specificity is achieved in this important post-translational modification. Since nitrosothiol formation requires a formal one-electron oxidation, a classification of reaction mechanisms is proposed regarding which species undergoes electron abstraction: NO, thiol or S-NO radical intermediate. Relevant kinetic, thermodynamic and mechanistic considerations will be examined and the impact of sources of NO and the chemical nature of potential reaction targets is also discussed.

Profile of urinary amino acids and their post-translational modifications (PTM) including advanced glycation end-products (AGEs) of lysine, arginine and cysteine in lean and obese ZSF1 rats

Heart failure with preserved ejection fraction (HFpEF) is associated with high mortality and has an increasing prevalence associated with the demographic change and limited therapeutic options. Underlying mechanisms are largely elusive and need to be explored to identify specific biomarkers and new targets, which mirror disease progression and intervention success. Obese ZSF1 (O-ZSF1) rats are a useful animal model, as they spontaneously develop hypertension, hyperlipidemia and glucose intolerance and finally HFpEF. The urinary profile of amino acids and their metabolites of post-translational modifications (PTM), including the advanced glycation end-products (AGEs) of lysine, arginine and cysteine, are poorly investigated in HFpEF and ZSF1 rats. The aim of the present study was to characterize the status of free amino acids and their metabolites of PTM and glycation in lean ZSF1 (L-ZSF1) and O-ZSF1 rats in urine aiming to find possible effects of glucose on the excretion of native and modified amino acids. In the urine of twelve L-ZSF1 and twelve O-ZFS1 rats collected at the age of 20 weeks, we measured the concentration of native and modified amino acids by reliable previously validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) approaches. Serum glucose was 1.39-fold higher in the O-ZSF1 rats, while urinary creatinine concentration was 2.5-fold lower in the O-ZSF1 rats. We observed many differences in urinary amino acids excretion between L-ZSF1 and O-ZSF1 rats. The creatinine-corrected homoarginine excretion was twofold lower in the O-ZSF1 rats. We also observed distinct associations between the concentrations of serum glucose and urinary amino acids including their PTM and AGE metabolites in the L-ZSF1 and O-ZSF1 rats. Our study shows that PTM metabolites and AGEs are consistently lower in the L-ZSF1 than in the O-ZSF1 rats. Serum malondialdehyde (MDA) concentration was higher in the O-ZSF1 rats. These results suggest that hyperglycemia, hyperlipidemia and elevated oxidative stress in the O-ZSF1 rats favor PTM methylation of arginine and lysine and the glycation of lysine and cysteine. The area under the receiver operation characteristic (ROC) curve values were 0.996 for serum glucose, 0.951 for urinary creatinine, 0.939 for serum MDA, 0.885 for Nε-carboxyethyl-lysine, 0.830 for carboxyethyl-cysteine, and 0.792 for monomethyl-lysine. Non-invasive measurement of methylation and glycation products of arginine, lysine and cysteine residues in proteins in urine of L-ZSF1 and O-ZSF1 rats may be useful in studying pathophysiology and pharmacology of HFpEF.

Abstract 1295: SJP1901, a small molecule inhibitor targeting hippo pathway by directly inhibiting TEAD palmitoylation in hippo pathway-dependent cancer

Abstract The hippo pathway is a critical transcriptional regulator of cell growth, proliferation, and migration in cancer, furthermore,YAP-TEAD complexes act as a major role in the pathway. S-palmitoylation of cysteine residues in TEAD proteins is required for their stability and function in hippo pathway signaling. Recently, it has become manifested that the dysregulation of hippo pathway effectors (MST1/2 and LATS1/2) is involved in oncogenesis. These effectors inactivate translocation of YAP and TAZ into the nucleus. The translocation of YAP/TAZ is responsible for acting as transcriptional coactivators by binding to the transcription factor, TEADs resulting in the transcription of cancer-related genes that drive tumor growth. Since YAP/TAZ are considered to be natively unfolded and can be difficult to handle as a drug target, TEAD is regarded as an excellent therapeutic target for intervention of the hippo pathway. SJP1901 is a small molecule of TEAD inhibitor with potential as drug candidates in malignant mesothelioma and other types of cancer. Based on the results of structure-based drug design, hit compounds were synthesized and their efficacy was evaluated.The compounds effectively inhibited palmitoylation of TEAD proteins and showed remarkable efficacy in reporter gene assay using TEAD responsive element (TRE)-integrated MCF7 with nano molar IC50 concentrations. The excellent anti-tumor effect of SJP1901 was revealed via cell proliferation inhibition assay (nano molar IC50 concentrations) andanalysis of inhibited transcription activity for target genes (CTGF and CYR61) usingmalignant mesothelia cell lines (NCI-H226, NCI-H2052 and MSTO-211H) known to display hippo pathway-dependent cell growth. In addition, these compounds showed lower toxicities in normal cells such as Fa2N4, CCD-18co and WI38 (IC50&amp;gt;10 μM). In conclusion, we obtained potent small molecules which inactivate TEAD proteins by directly inhibiting their palmitoylation. SJP1901 showed outstanding anti-cancer effects through regulating hippo pathway-mediated target genes and lower toxicitiy in normal cells. In vivo efficacy study and research to expand the indications are currently ongoing. Citation Format: Jihyun Um, Janghyun Lee, Kwangwoo Hwang, Sujin Park, Jooyoung Hyun, Dohyeong Lee, Jeongmin Lee, Li-Kyung Kim, Moon Jung Back, Seong Jun Park, Hwan Jung Lim, You-Keun Shin, Hei-Cheul Jeung, Jaewoong Lee, Hyun Tae Kim, Yongbin Park, Hoseok Kwon, Min-Hyo Ki. SJP1901, a small molecule inhibitor targeting hippo pathway by directly inhibiting TEAD palmitoylation in hippo pathway-dependent cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1295.

Glutathione conjugation and protein modification resulting from metabolic activation of venlafaxine in vitro and in vivo

Venlafaxine (VLF), an antidepressant agent, is widely used to combat major depressive disorders, particularly for the treatment of selective serotonin reuptake inhibitor-resistant depression. VLF has been shown to cause liver injury. The present study aimed to investigate the metabolic activation of VLF and explore the mechanisms of hepatotoxicity induced by VLF. One glutathione (GSH) conjugate and one cysteine conjugate were both detected in mouse and human liver microsomal incubations containing VLF and GSH or cysteine. The two conjugates were also detected in cultured mouse primary hepatocytes and bile of rats after exposure to VLF. The in vitro and in vivo studies demonstrated that VLF was metabolized to a quinone methide intermediate reactive to GSH and cysteine residues of hepatic protein. The observed protein covalent binding revealed dose-dependency. The metabolic activation of VLF was P450-dependent, and CYP3A4 was found as the predominant enzyme involved in the bioactivation process. These findings facilitate better understanding of the metabolic activation-hepatotoxicity relationship of VLF and provide chemists with information about new potential structural alerts during drug design process.