Biol Chem Research Articles

ENL mutation and AML: a new model that reveals oncogenic condensate's function in leukemogenesis.

Precise regulation of gene expression is essential for proper development and the maintenance of homeostasis in organisms. Studies have shown that some transcriptional regulatory proteins influence gene expression through the formation of dynamic, locally concentrated assemblies known as condensates, while dysregulation of transcriptional condensates was associated with several cancers, such as Ewing sarcoma and AML [Wang Y etal. (2023) Nat Chem Biol 19, 1223-1234; Chandra B etal. (2022) Cancer Discov 12, 1152-1169]. Mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL) have been shown to form discrete condensates at endogenous genomic targets, but it remains unclear how ENL mutations drive tumorigenesis and whether it is correlated with their condensate formation property. Liu etal. now show, using a conditional knock-in mouse model, that ENL YEATS domain mutation is a bona fide oncogenic driver for AML. This mutant ENL forms condensates in hematopoietic stem/progenitor cells at the genomic loci of key leukemogenic genes, including Meis1 and Hoxa cluster genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Furthermore, they show that small-molecule inhibition of the acetyl-binding activity displaces ENL mutant condensates from oncogenic target loci, and this inhibitor significantly impairs the onset and progression of AML driven by mutant ENL in vivo.

Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution.

Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559-31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

A methylene blue assay for the quantification of fucoidan – A specific application for seaweed extracts

Fucoidan is a fucose-rich sulfated polysaccharide found mainly in brown seaweeds. Fucoidan has recently gained much attention due to its bioactive and medicinal properties, hence its high potential to be utilized in the pharmaceutical industry. Therefore, an accurate, reliable, and simple method for the specific determination of fucoidan would be a useful tool when looking to recover this valuable polysaccharide. This research focused on developing an assay utilizing the thiazine dye, methylene blue, for the quantification of fucoidan, with a specific application for fucoidan found in algal extracts. Fucoidan could be specifically determined at pH 1, to maximum concentrations of 4 g L-1 in solution without the interference of alginate and other seaweed extract contaminants. At pH 7 and 11, quantification of fucoidan led to interference from alginate and gallic acid. A conversion factor of 1.8 allowed the quantification of fucoidan from Ecklonia maxima extracts using a commercial standard from Fucus vesiculosis. An Ecklonia maxima extract was found to contain 5.47 ± 0.061 g L-1 of fucoidan. Measuring the fucoidan and L-fucose concentration in crude seaweed extract through the methylene blue assay and modified Dische and Shettles J Biol Chem 175:595–603, (1948) method, respectively, confirmed the accuracy and specificity of the method. The limit of quantification of the methylene blue assay at pH 1 was 0.62 g L-1 of fucoidan.

Activation of the Human Epithelial Sodium Channel (ENaC) by the Peptidomimetic S3969 Involves a Specific Binding Pocket in the Channel’s β-Subunit

The epithelial sodium channel (ENaC) mediates Na+ absorption in several epithelia. Its impaired function leads to severe disorders, like pseudohypoaldosteronism type 1 and respiratory distress. Interestingly, a small molecule ENaC activator, the peptidomimetic S3969, stimulates human but not mouse αβγ-ENaC (Lu et al. 2008, J Biol Chem). Our aim was to identify and characterize the S3969 binding site in human ENaC. ENaC was heterologously expressed in Xenopus laevis oocytes, amiloride-sensitive whole-cell currents (ΔIami) were measured using the two-electrode voltage clamp technique. A putative S3969 binding site was predicted by using molecular docking and molecular dynamics (MD) simulations based on a recently published ENaC structure (Noreng et al. 2020, Elife). In addition, the effect of S3969 on endogenously expressed ENaC was studied in human airway epithelial cells (H441) using transepithelial measurements in Ussing chambers. We confirmed that in oocytes S3969 activated human αβγ-ENaC by ~2-fold with an EC50 of ~0.3 μM (n=25). As reported, murine αβγ-ENaC was insensitive to S3969 in concentrations up to 10 μM (n=19). Using mouse-human chimeric ENaC, we found that a portion of the extracellular loop of the β-subunit (residues 256-404), which includes parts of the thumb, palm and β-ball domains, is critical for channel stimulation by S3969. Computer simulations predicted a putative S3969 binding pocket in this area, with βR388, βF391, and βY406 as key residues coordinating S3969. Mutating these residues strongly reduced (βR388A) or nearly abolished (βY406A; βF391G) the stimulatory effect of S3969 on ΔIami (n=12-18), without altering the effect of an alternative ENaC activator chymotrypsin (n=5). MD simulations also suggested that the binding of S3969 to ENaC increased the distance between the β-thumb and the γ-palm domain. Consistent with this, the stimulatory effect of S3969 was abolished when the β-thumb domain was covalently attached to the γ-palm domain following the introduction of two cysteine residues (βR437C, γS298C) to form a disulfide bridge (n=15). Importantly, reducing the disulfide bond with DTT partially rescued the effect of S3969 on ΔIami (relative stimulation 31±1%, n=15, p<0.001). We also showed that S3969 stimulated endogenously expressed human ENaC in H441 cells. In conclusion, we demonstrated that S3969 activates ENaC by interacting with a specific binding pocket in β-ENaC which probably increases the β-γ-intersubunit distance. These results may help to identify novel ENaC modulators with potential physiological or therapeutic implications. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 509149993, TRR 374 (subproject A4 to A.I. and C.K.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hemicyanine-Phenothiazine Based Highly Selective Ratiometric Fluorescent Probes for Detecting Hypochlorite Ion in Fruits, Vegetables and Beverages.

Hypochloric acid (HClO) is a reactive oxygen species (ROS) that functions as a bacteriostatic and disinfectant in food production. Excessive levels of ClO-, however, have been linked to various health issues, including cardiovascular diseases (Halliwell and Gutteridge in Oxford University press, USA, 2015), arthritis, and neurodegenerative diseases (Heinzelmann and Bauer in Biol Chem. 391(6):675-693, 2010). Therefore, synthesizing highly selective and sensitive probes for rapidly detecting endogenous ClO- in daily foods is currently a popular research topic (Kalyanaraman et al. in Redox Biol. 15:347-362, 2018;Winterbourn in Nat Chem Biol. 4(5):278-286, 2008; Turrens in J Physiol. 552(2):335-344, 2003). Thus, we have developed two highly selective ratiometric fluorescent probes (Probe1 and Probe2) based on indole-phenothiazine to detect ClO- in common vegetables, fruits and beverages qualitatively and quantitatively. Moreover, Both Probe1 and Probe2 have shown good specificity and stability, with high fluorescence intensity and long duration (Feng et al. in Adv Sci. 5:1800397, 2018; Wei et al. in Angew Chem. 131(14):4595-4599, 2019; Baruah et al. in J Mater Chem B, 2022).

Studying RAS Interactions in Live Cells with BRET.

Bioluminescence resonance energy transfer (BRET) is a valuable technique for studying protein-protein interactions (PPIs) within live cells (Pfleger and Eidne, Nat Methods 3:165-174, 2006). Among the various BRET methodologies, a recent addition called NanoBRET has emerged, leveraging advancements in donor and acceptor technologies (Machleidt and Woodroofe, ACS Chem Biol 10:1797-1804, 2015). In this study, we present a developed methodology designed to measure PPIs involving the RAS protein family and their effectors and interactors at the plasma membrane. By utilizing the NanoLuc and HaloTag BRET pair, we provide evidence of a saturable interaction between KRAS4b-G12D and full-length RAF1. Conversely, the RAF1 R89L mutant, known to impede RAF1 binding to active RAS, exhibits nonspecific interactions. The assay exhibits remarkable signal-to-background ratios and is highly suitable for investigating the interactions of RAS with effectors, as well as for high-throughput screening assays.

Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1′-position amino acid residue

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2′-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1′ and P2′ residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1′-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1′ residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1′ amino acid residues.

Expression and gene regulatory network of S100A16 protein in cervical cancer cells based on data mining

S100A16 protein belongs to the S100 family of calcium-binding proteins, which is widely distributed in human tissues and highly conserved. S100 calcium-binding proteins possess broad biological functions, such as cancer cell proliferation, apoptosis, tumor metastasis, and inflammation (Nat Rev Cancer 15:96–109, 2015). The S100A16 protein was initially isolated from a cell line derived from astrocytoma. The S100A16 protein, consisting of 103 amino acids, is a small acidic protein with a molecular weight of 11,801.4 Da and an isoelectric point (pI) of 6.28 (Biochem Biophys Res Commun 313:237–244, 2004). This protein exhibits high conservation among mammals and is widely expressed in various human tissues (Biochem Biophys Res Commun 322:1111–1122, 2004). Like other S100 proteins, S100A16 contains two EF-hand motifs that form a helix-loop-helix structural domain. The N-terminal domain and the C-terminal domain of S100A16 are connected by a "hinge" linker.S100A16 protein exhibits distinct characteristics that distinguish it from other S100 proteins. A notable feature is the presence of a single functional Ca2 + binding site located in the C-terminal EF-hand, consisting of 12 amino acids per protein monomer (J Biol Chem 281:38905–38917, 2006). In contrast, the N-terminal EF-hand of S100A16 comprises 15 amino acids instead of the typical 14, and it lacks the conserved glutamate residue at the final position. This unique attribute may contribute to the impaired Ca2 + binding capability in the N-terminal region (J Biol Chem 281:38905–38917, 2006). Studies have shown an integral role of S100 calcium-binding proteins in the diagnosis, treatment, and prognosis of certain diseases (Cancers 12:2037, 2020). Abnormal expression of S100A16 protein is implicated in the progression of breast and prostate cancer, but an inhibitor of oral cancer and acute lymphoblastic leukemia tumor cell proliferation (BMC Cancer 15:53, 2015; BMC Cancer 15:631, 2015). Tu et al. (Front Cell Dev Biol 9:645641, 2021) indicate that the overexpression of S100A16 mRNA in cervical cancer(CC) such as cervical squamous cell carcinoma and endocervical adenocarcinoma as compared to the control specimens. Tomiyama N. and co-workers (Oncol Lett 15:9929–9933, 2018) (Tomiyama, N) investigated the role of S100A16 in cancer stem cells using Yumoto cells (a CC cell line),The authors found upregulation of S100A16 in Yumoto cells following sphere formation as compared to monolayer culture.Despite a certain degree of understanding, the exact biological function of S100A16 in CC is still unclear. This article explores the role of S100A16 in CC through a bioinformatics analysis. Referencing the mRNA expression and SNP data of cervical cancer available through The Cancer Genome Atlas (TCGA) database, we analyzed S100A16 and its associated regulatory gene expression network in cervical cancer. We further screened genes co-expressed with S100A16 to hypothesize their function and relationship to the S100A16 cervical cancer phenotype.Our results showed that data mining can effectively elucidate the expression and gene regulatory network of S100A16 in cervical cancer, laying the foundation for further investigations into S100A16 cervical tumorigenesis.

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P <0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

SAT014 Involvement Of Cytochrome P450 1B1 In Synthesis Of Neurosteroid Pregnenolone In Human Brain Cells

Abstract Disclosure: Y. Lin: None. G. Cheung: None. Z. Zhang: None. V. Papadopoulos: None. Neurosteroids, steroids synthesized in the nervous system, have important functions in modulating brain function, neuroprotection, and reducing neuroinflammation. The neurosteroid pregnenolone, the precursor to all other neurosteroids, is one of the most abundant neurosteroids in the brain. Still, the enzyme known to make it, CYP11A1, has been difficult to detect in the human brain. We recently reported that unlike in peripheral steroidogenic organs, pregnenolone is not synthesized by CYP11A1 in human brain cells but rather by another mitochondrial cytochrome P450 enzyme (J Biol Chem. 2022 Jul;298(7):102110). Therefore, we aimed to identify the enzyme responsible for producing pregnenolone in the current study. Upon bioinformatic analyses of publicly available datasets for mitochondrial CYP450s expressed in the human brain, we identified three potential candidates: CYP27A1, CYP1A1, and CYP1B1. Given that CYP27A1 is highly expressed in the brain and is involved in cholesterol metabolism, we first tested the effects of CYP27A1 inhibition via drug treatment and siRNA knockdown on pregnenolone synthesis in steroidogenic MGM-1 human glioma cells. Neither treatment with CYP27A1 inhibitors anastrozole or dexmedetomidine nor CYP27A1 siRNA knockdown resulted in a significant reduction in pregnenolone formation by MGM-1 cells, suggesting that CYP27A1 likely is not involved in pregnenolone synthesis in human brain cells. On the other hand, MGM-1 cells treated with the CYP1A1/CYP1B1 inhibitors α-napthoflavone and 2,4,3’,5’-tetramethoxystilbene showed a significant reduction in pregnenolone production in the presence of the pregnenolone precursor 22R-hydroxycholesterol. To investigate whether this effect was due to the inhibition of CYP1A1 or CYP1B1, we knocked down CYP1A1 or CYP1B1 in MGM-1 cells using siRNA. While CYP1A1 knockdown did not result in any significant changes to pregnenolone synthesis, CYP1B1 knockdown significantly decreased secreted pregnenolone formation by MGM-1 cells. Together, these results suggest that CYP1B1 is likely involved in the alternative pathway for pregnenolone synthesis in human brain cells. Presentation: Saturday, June 17, 2023

FRI669 Skeletal Effect Of Cyclic Guanosine Monophosphate Dependent Protein Kinase G Type 2

Abstract Disclosure: S. Kim: None. E. Shelly: None. F. Sultana: None. F. Korkmaz: None. M. Temple: None. J. Gimenez: None. V. Muradova: None. T. Yuen: None. M. Zaidi: None. The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a significant role in bone remodeling. PKG type 2 (PKG2) is expressed in bone cells [1-2], and mice with osteoblast-specific PKG2 upregulation (Col1a1-Prkg2R242Q) showed increased bone formation and preserved bone in mice with streptozotocin-induced diabetes [3]. In this study, we have crossed Prkg2fl/fl mice with Col2.3-Cre mice and Acp5-Cre mice, respectively, to generate osteoblast-specific and osteoclast-specific Prkg2 knockout mutants. The areal BMD of osteoblast-specific or osteoclast-specific mice were not different from the respective Cre- controls at 4, 6 and 8 months. µCT did not show any difference in fractional bone volume (BV/TV), or trabecular parameters compared with the Cre- controls. However, when mice were put under skeletal insults through glucocorticoid injection (dexamethasone, 10 mg/kg, SQ, daily, 4 month), Col2.3-Cre+;Prkg2fl/+ mice lost more bone compared with Col2.3-Cre−;Prkg2fl/+ mice at the tibia (−23.1% vs. −12.1%, P=0.011). In addition, we observed the osteoblast-specific Prkg2 knockout mice displayed increased body weight (Col2.3-Cre+;Prkg2fl/fl +25.1%**, Col2.3-Cre+;Prkg2fl/+ +19.9%*, Col2.3-Cre−;Prkg2fl/fl +7.3%), increased fat mass (Col2.3-Cre+;Prkg2fl/fl +26.0%*, Col2.3-Cre+;Prkg2fl/+ +21.4%**, Col2.3-Cre−;Prkg2fl/fl −3.3%), and decreased lean mass (Col2.3-Cre+;Prkg2fl/fl −4.3%*, Col2.3-Cre+;Prkg2fl/+ −2.9%, Col2.3-Cre-;Prkg2fl/fl +0.3%) from the baseline (**P < 0.01 and *P < 0.05 vs. Cre- controls). Furthermore, we noted a longer femur length in the osteoblast-specific Prkg2 knockout mutants. At 8 months, the femur length of Col2.3-Cre+;Prkg2fl/fl and Col2.3-Cre+;Prkg2fl/+ were 9.3% (P < 0.01) and 7.5% (P = 0.01), respectively, longer than the Cre- controls. Our findings suggest that PKG2 is necessary for protecting bone from glucocorticoid insults and abolishing PKG2 in osteoblasts might affect the differentiation of mesenchymal stem cell towards adipocytes and chondrocytes at the expense of osteoblasts and myocytes. [1] Kim et al, Ann N Y Acad Sci, 2021[2] Rangaswami J et al, Biol Chem. 2009[4] Ramdani et al, J Endocrinol 2018 Presentation: Friday, June 16, 2023

Fortuitous In Vitro Compound Degradation Produces a Tractable Hit against Mycobacterium tuberculosis Dethiobiotin Synthetase: A Cautionary Tale of What Goes In Does Not Always Come Out.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here, the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by an in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD = 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here, we show that the lactone of 1 hydrolyzes to a carboxylic acid (2) under basic conditions, including those of the crystallography soak, with a subsequent ring opening of the component cyclobutane ring forming a cyclopentylacetic acid (3). Crystals soaked directly with authentic 3 produced an electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled the subsequent compound development of nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.

Vitamin C: Novel Functions in Bone Homeostasis

Vitamin C is best known for its essential role in collagen biosynthesis and maturation and in maintaining the integrity of connective tissues, including bone. Recent studies reported in influential journals have identified some novel functions of vitamin C in bone biology, including controlling osteoblastic differentiation and bone mineralization. These actions of vitamin C in regulating bone homeostasis may make it a potentially effective agent for osteoporosis intervention. (First online: June 4, 2023) REFERENCES Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnell SR. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci USA 1981; 78(5):2879–82. doi: https://dx.doi.org/10.1073/pnas.78.5.2879 Gabbay KH, Bohren KM, Morello R, Bertin T, Liu J, Vogel P. Ascorbate synthesis pathway: dual role of ascorbate in bone homeostasis. J Biol Chem 2010; 285(25):19510–20. doi: https://dx.doi.org/10.1074/jbc.M110.110247 Thaler R, Khani F, Sturmlechner I, Dehghani SS, Denbeigh JM, Zhou X, et al. Vitamin C epigenetically controls osteogenesis and bone mineralization. Nat Commun 2022; 13(1):5883. doi: https://dx.doi.org/10.1038/s41467-022-32915-8 Malmir H, Shab-Bidar S, Djafarian K. Vitamin C intake in relation to bone mineral density and risk of hip fracture and osteoporosis: a systematic review and meta-analysis of observational studies. Br J Nutr 2018; 119(8):847–58. doi: https://dx.doi.org/10.1017/S0007114518000430 Compston JE, McClung MR, Leslie WD. Osteoporosis. Lancet 2019; 393(10169):364–76. doi: https://dx.doi.org/10.1016/S0140-6736(18)32112-3 Collaborators GBDF. Global, regional, and national burden of bone fractures in 204 countries and territories, 1990–2019: a systematic analysis from the Global Burden of Disease Study 2019. Lancet Healthy Longev 2021; 2(9):e580–e92. doi: https://dx.doi.org/10.1016/S2666-7568(21)00172-0

Abstract B051: Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC

Abstract Background: Histone modifying enzymes (HMEs) hold significant promise as targets for drug discovery in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) dictate the presence of acetyl marks, which regulate the openness of chromatin and transcriptional activation. We previously developed a novel histone array on a chip to measure HME dysregulation with the development of castration resistance (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC. This alteration was subsequently identified in a subset of castration-resistant tumors (BMC Cancer 2017). The goal of the current study was to determine if the isolation of circulating tumor cells (CTCs) could be used as a biomarker to identify patients with altered p300 acetylation activity. Methods: Peripheral blood was collected from 13 patients (15 samples) with advanced PC after informed written consent under the University of Wisconsin IRB-approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using pre-conjugated, anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled Sera-Mag streptavidin-coated magnetic beads (SM). Cells bound to SMs were isolated with ESP technology using an automated magnetic PipetMax pipetting robot. SM-bound cells were moved through staining and washing wells prior to imaging at 20x with a Nikon Eclipse Ti fluorescent microscope and NIS-Elements AR 4.10 software, and finally images were analyzed with NIS Elements software. Results: We first optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in prostate cancer cell lines. Employing a selective inhibitor of p300 acetylation (A-485) reduced levels of p300 and H3K18 acetylation. We then confirmed that CTCs from patient samples were successfully captured by the EpCAM antibody and show clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Increased acetylation of p300 and H3K18 and decreased SIRT2 is found in 6/10 CRPC samples compared to HSPC patients. Paired samples collected from one patient show a marked increase in p300 activity, H3K18 hyperacetylation, and a decrease in SIRT2 expression in the later developed CRPC sample compared to the HSPC sample. Another CRPC subject, decreased acetyl p300 and histone acetyl-H3k18, and increased SIRT2 staining is noted several months after beginning enzalutamide administrated. Analysis in CTCs indicates a positive correlation between acetyl-p300 and H3K18ac (R=0.61) and between SIRT2 and H3K18ac (R=-0.59). Conclusions: This study suggests that acetyl-p300, H3K18ac, and SIRT2 are epigenetic biomarkers that increase in a subset of CTC from CRPC. These biomarkers could potentially be used for monitoring outcomes and predicting tumor responses to developing p300 acetylation inhibitors. Citation Format: Mikolaj Filon, Bing Yang, Jennifer Schehr, Anupama Singh, Marcelo Bigarella, John Denu, Joshua Lang, David Jarrard. Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B051.

Re‐evaluation of the genetic role of a rare APOE variant (L28P; APOE*4Pittsburgh) in late‐onset Alzheimer disease

AbstractBackgroundA rare missense APOE variant (L28P; APOE*4Pittsburgh) has been reported to be a risk factor for Alzheimer’s disease (AD). However, sinceL28P has been observed only among APOE*4 carriers, its independent genetic association is uncertain. In this study, we re‐evaluated this association in a large case‐control sample of 15,762 U.S. Whites aged ≥60 years and investigated its independent effect.MethodSamples were derived from three sources: University of Pittsburgh, Alzheimer’s Disease Sequencing Project and the Gingko Evaluation Memory Study. Due to variation in the age distribution between cases and controls in the three studies, each study sample was analyzed separately, and the results then combined by meta‐analysis. To distinguish the independent effect of L28P from APOE*4, we restricted the analysis to subjects with the APOE 3/4 genotype, as L28P has been observed only in the heterozygous state in the APOE*4‐background and 3/4 is the most common genotype containing the APOE*4 allele.ResultA total of 80 L28P heterozygotes were observed in the combined case‐control sample, all in those containing only the APOE*4 allele, confirming the complete linkage disequilibrium between the two sites. The age‐ and sex‐adjusted meta‐analysis odds ratio (OR) was 2.87 (95% CI: 1.34 – 6.13; p = 0.0066). There were a total of 4,138 cases and controls with the 3/4 genotype. The age‐ and sex‐adjusted meta‐analysis OR was 1.53 (95% CI: 0.70 – 3.36; p = 0.28). The lack of significance is mainly due to the low power with the ∼4,100 sample size (12% power at α = 0.80), as compared to the required sample size of ∼151,000 to detect an OR of 1.5 at α = 0.80.ConclusionEven with non‐significant p‐value, the OR of 1.53 among 3/4 subjects suggests that the effect of L28P on AD risk is independent of APOE*4. Our genetic finding is reinforced by an earlier experimental finding showing that this mutation leads to significant structural and conformational alterations in ApoE and can induce functional defects associated with neuronal Aβ42 accumulation and oxidative stress (J Biol Chem 2014; 289:12931). Additional studies in cell‐based systems and animal models will help to delineate its functional significance in the AD etiology.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( > 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

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Chemical Biology & Drug DesignVolume 101, Issue 6 p. i-iii ISSUE INFORMATIONFree Access Cover Image First published: 11 May 2023 https://doi.org/10.1111/cbdd.14078AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover artwork: Boosting Pose Ranking Performance via Rescoring with MM-GBSA. Chem Biol Drug Des 88:317–328. Volume101, Issue6June 2023Pages i-iii RelatedInformation

Dieter Wolf (1941–2023): a life dedicated to understanding protein quality control and the ubiquitin‐proteasome system

Dieter Wolf (1941–2023): a life dedicated to understanding protein quality control and the ubiquitin‐proteasome system

In mouse kidney endogenous transmembrane serine protease 2 (TMPRSS2) contributes to proteolytic processing and activation of the epithelial sodium channel (ENaC)

Regulation of ENaC in the distal nephron is essential for sodium homeostasis and blood pressure control. A unique feature of ENaC is its complex proteolytic processing leading to channel activation due to release of inhibitory tracts from its α- and γ-subunits. In particular, fully cleaved γ-ENaC is critical for high channel activity. However, the physiologically relevant proteases involved remain elusive. Using Xenopus laevis oocytes and H441 airway epithelial cells, we recently demonstrated that TMPRSS2 proteolytically activates ENaC by cleaving the channel’s γ-subunit ( J Biol Chem 2022 Jun;298(6):102004). Here we investigate whether TMPRSS2 also contributes to proteolytic ENaC regulation in the kidney. Using mRNA sequencing, we demonstrated that mouse cortical collecting duct (mCCD cl1 ) cells express TMPRSS2 at a high level unaffected by aldosterone, an important ENaC stimulating hormone. TMPRSS2 knockdown by CRISPR/Cas9 technology significantly reduced fully cleaved γ-ENaC in membrane-enriched fractions of mCCD cl1 cell lysates (n=5). Apical application of chymotrypsin significantly stimulated ENaC-mediated short circuit current ( I SC ) in TMPRSS2-knockdown cells, but not in control cells (2.0 ± 1.1 μA/cm² vs. 0.2 ± 0.6 μA/cm²; n=13-14; p<0.001; mean ± SD). Moreover, the stimulatory effect of aldosterone on I SC was impaired in TMPRSS2-knockdown cells compared to control cells (9.1 ± 4.0 μA/cm² vs. 18.3 ± 4.0 μA/cm²; n=7-8; p<0.01). After aldosterone treatment I SC stimulation by chymotrypsin was enhanced in TMPRSS2-knockdown cells (5.3 ± 1.9 μA/cm²; n=7) but undetectable in control cells (−0.8 ± 1.6 μA/cm²; n=8). These findings indicate that TMPRSS2 deficiency impairs proteolytic ENaC activation in mCCD cl1 cells. To confirm this in an in vivo model, we used constitutive TMPRSS2 knockout mice ( Tmprss2 -/- ). Using a combination of RNAscope technology and immunofluorescence, we demonstrated high expression of Tmprss2 mRNA in renal tubular cells, including cells with β-ENaC protein staining. Importantly, the ratio of fully to partially cleaved γ-ENaC was significantly reduced in membrane-enriched fractions of whole-kidney lysates from Tmprss2 -/- mice (n=10). Maintained on a standard diet, Tmprss2 -/- mice had no apparent renal phenotype with plasma aldosterone levels similar to those of Tmprss2 +/+ mice (120 ± 43 pg/ml in Tmprss2 -/- vs. 92 ± 17 pg/mL in Tmprss2 +/+ ; n=7-10). Moreover, the ability of Tmprss2 -/- mice to reduce urinary sodium excretion in response to four days of low sodium diet was fully preserved. However, under low sodium diet plasma aldosterone increased to significantly higher levels in Tmprss2 -/- mice compared to controls (752 ± 146 pg/ml in Tmprss2 -/- vs. 268 ± 55 pg/ml in Tmprss2 +/+ ; n=7-11; p<0.01). This is most likely a compensatory response to compromised proteolytic ENaC activation due to TMPRSS2 deficiency. We conclude that TMPRSS2 plays a physiological role in proteolytic ENaC processing and activation in the kidney. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 509149993, TRR 374 (subproject A4 to A.I. and C.K.) and the Bayerisches Staatsministerium für Wissenschaft und Kunst (Bavarian Ministry of Art and Science), project VI-Corona-Forschung (subproject 07 to M.B. and T.G). A.K. was supported by the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Erlangen. S.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education and Research, Germany), project SENSE-CoV2, 01KI20172A. P.S. and F.A. were funded by the IZKF Tübingen. The authors declare no conflicts of interest. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.