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The stereochemical properties of N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones (2a–c), which inhibit potassium channels in T cells, were examined by freezing their conformational change due to 4-methyl substitution. N-Acyl-5H-dibenzo[b,d]azepin-7(6H)-ones exist as pairs of enantiomers [(a[sup.1]R, a[sup.2]R), (a[sup.1]S, a[sup.2]S)], and each atropisomer is separable at room temperature. An alternate procedure for preparing 5H-dibenzo[b,d]azepin-7(6H)-ones involves the intramolecular Friedel–Crafts cyclization of N-benzyloxycarbonylated biaryl amino acids. Consequently, the N-benzyloxy group was removed during the cyclization reaction to produce 5H-dibenzo[b,d]azepin-7(6H)-ones suitable for the subsequent N-acylation reaction.

Summary Drought is a major abiotic stress that threatens maize production globally. A previous genome‐wide association study identified a significant association between the natural variation of ZmTIP1 and the drought tolerance of maize seedlings. Here, we report on comprehensive genetic and functional analysis, indicating that ZmTIP1, which encodes a functional S‐acyltransferase, plays a positive role in regulating the length of root hairs and the level of drought tolerance in maize. We show that enhancing ZmTIP1 expression in transgenic Arabidopsis and maize increased root hair length, as well as plant tolerance to water deficit. In contrast, ZmTIP1 transposon‐insertional mutants displayed the opposite phenotype. A calcium‐dependent protein kinase, ZmCPK9, was identified as a substrate protein of ZmTIP1, and ZmTIP1‐mediated palmitoylation of two cysteine residues facilitated the ZmCPK9 PM association. The results of this research enrich our knowledge about ZmTIP1‐mediated protein S‐acylation modifications in relation to the regulation of root hair elongation and drought tolerance. Additionally, the identification of a favourable allele of ZmTIP1 also provides a valuable genetic resource or selection target for the genetic improvement of maize.

The smallest histone deacetylase (HDAC) and the only class IV HDAC member, HDAC11, is reported to regulate immune activation and tumorigenesis, yet its biochemical function is largely unknown. Here we identify HDAC11 as an efficient lysine defattyacylase that is >10,000-fold more efficient than its deacetylase activity. Through proteomics studies, we hypothesized and later biochemically validated SHMT2 as a defatty-acylation substrate of HDAC11. HDAC11-catalyzed defatty-acylation did not affect the enzymatic activity of SHMT2. Instead, it affects the ability of SHMT2 to regulate type I IFN receptor ubiquitination and cell surface level. Correspondingly, HDAC11 depletion increased type I IFN signaling in both cell culture and mice. This study not only demonstrates that HDAC11 has an activity that is much more efficient than the corresponding deacetylase activity, but also expands the physiological functions of HDAC11 and protein lysine fatty acylation, which opens up opportunities to develop HDAC11-specific inhibitors as therapeutics to modulate immune responses.

In mouse and humans, the X-chromosomal Porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands, thus representing a bottleneck in the secretion of Wnt ligands. In humans, mutations in PORCN cause the X-linked dominant syndrome Focal Dermal Hypoplasia (FDH, OMIM#305600). This disorder is characterized by ecto-mesodermal dysplasias and shows a highly variable phenotype, potentially due to individual X chromosome inactivation patterns. To improve the understanding of human FDH, we have established a mouse model by generation of Porcn heterozygous animals carrying a zygotic deletion of the paternal allele. We show that heterozygous female fetuses display variable defects that do not significantly affect survival in the uterus, but lead to perinatal lethality in more than 95% of females. Rare survivors develop to adulthood and display variable skeletal and skin defects, representing an adult zygotic mouse model for human FDH. Although not frequently reported in humans, we also observed bronchopneumonia, rhinitis, and otitis media in these animals, suggesting a potential link between Porcn function and the normal development of ciliated cells in these tissues.

A transition metal-free synthesis of 1,2,5-trisubstituted 1H-indoles by a deprotonation–S[sub.N]Ar cyclization sequence from 1-aryl-2-(2-fluoro-5-nitrophenyl)ethan-1-one (deoxy-benzoin) Schiff bases is reported. The starting deoxybenzoins were prepared by Friedel-Crafts acylation of activated aromatic compounds by 2-(2-fluoro-5-nitrophenyl)acetyl chloride with AlCl[sub.3] or the corresponding acid with (CH[sub.3]SO[sub.2])[sub.2]O. The Schiff bases were generated by slow distillation of toluene (18–24 h) from a heated solution of each deoxybenzoin (1 equiv) with a benzyl- or phenethylamine, a high-boiling aliphatic amine, or an aniline derivative (5 equiv). Subsequent addition of N,N-dimethylformamide, 2 equiv of anhydrous K[sub.2]CO[sub.3], and heating at 90–95 °C for 18–24 h completed the synthesis. Benzyl-, phenethyl-, and high-boiling amines gave excellent yields while the heating requirements for the initial condensation made volatile aliphatic amines difficult to use and gave low yields. Aniline reactivities correlated with substituent-derived base strength, although modified conditions allowed some yields to be improved. Several anticipated competing processes had minimal impact on the outcome of the cyclizations.

A novel cationic microbubble (MB) for improvement of the DNA loading capacity and the ultrasound-mediated gene delivery efficiency has been developed; it has been prepared with commercial lipids and a stearic acid modified polyethylenimine 600 (Stearic-PEI600) polymer synthesized via acylation reaction of branched PEI600 and stearic acid mediated by N, N'-carbonyldiimidazole (CDI). The MBs' concentration, size distribution, stability and zeta potential ( zeta -potential) were measured and the DNA loading capacity was examined as a function of the amount of Stearic-PEI600. The gene transfection efficiency and cytotoxicity were also examined using breast cancer MCF-7 cells via the reporter plasmid pCMV-Luc, encoding the firefly luciferase gene. The results showed that the Stearic-PEI600 polymer caused a significant increase in magnitude of zeta -potential of MBs. The addition of DNA into cationic MBs can shift zeta -potentials from positive to negative values. The DNA loading capacity of the MBs grew linearly from (5 plus or minus 0.2) 10-3 pg/ mu m2 to (20 plus or minus 1.8) 10-3 pg/ mu m2 when Stearic-PEI600 was increased from 5 mol% to 30 mol%. Transfection of MCF-7 cells using 5% PEI600 MBs plus ultrasound exposure yielded 5.76 plus or minus 2.58103 p/s/cm2/sr average radiance intensity, was 8.97- and 7.53-fold higher than those treated with plain MBs plus ultrasound (6.41 plus or minus 5.82) 102 p/s/cm2/sr, (P<0.01) and PEI600 MBs without ultrasound (7.65 plus or minus 6.18) 102 p/s/cm2/sr, (P<0.01), respectively. However, the PEI600 MBs showed slightly higher cytotoxicity than plain MBs. The cells treated with PEI600-MBs and plain MBs plus ultrasound showed 59.5 plus or minus 6.1% and 71.4 plus or minus 7.1% cell viability, respectively. In conclusion, our study demonstrated that the novel cationic MBs were able to increase DNA loading capacity and gene transfection efficiency and could be potentially applied in targeted gene delivery and therapy.

Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if b > 1 there is substrate activation, if b < 1 there is substrate inhibition. The mechanistic significance of the b factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines b. However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that b depends on the acylation step. The magnitude of b and opposite b values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the Ω and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.

Background Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT) of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP), and C5a, involved in innate immunity. Aim We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl) and C5L2 knock-out (C5L2-/-) mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO) conditions. Results In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold) both over time and with DIO. By contrast, in C5L2-/-, there was no change in C5aR in WAT. C5L2-/- mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro-) vs M2 (anti-inflammatory) macrophage proportion was unchanged but C5L2-/- adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2-/- mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2-/- mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance. Conclusion Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis 1 – 3 , and aberrant Wnt signalling is frequently associated with cancers 4 . Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O -acyltransferase Porcupine 5 – 7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling 8 , 9 . Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials 10 ; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment. Cryo-electron microscopy structures of human Porcupine in complex with palmitoleoyl-coenzyme A, the inhibitor LGK974 and its peptide substrate suggest a mechanism for Wnt acylation.