A sterol analog inhibits hedgehog pathway by blocking cholesterylation of smoothened.

The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.

GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum.

Hedgehog (Hh) signaling pathway plays a pivotal role in embryonic development. Hh binding to Patched1 (PTCH1) derepresses Smoothened (SMO), thereby activating the downstream signal transduction. Covalent SMO modification by cholesterol in its cysteine-rich domain (CRD) is essential for SMO function. SMO cholesterylation is a calcium-accelerated autoprocessing reaction, and STIM1-ORAI1-mediated store-operated calcium entry promotes cholesterylation and activation of endosome-localized SMO. However, it is unknown whether the Hh-PTCH1 interplay regulates the activity of the endoplasmic reticulum (ER)-localized SMO. Here, we found that PTCH1 inhibited the COPII-dependent export of SMO from the ER, whereas Hh promoted this process. The RRxWxR amino acid motif in the cytosolic tail of SMO was essential for COPII recognition, ciliary localization, and signal transduction activity. Hh and PTCH1 regulated cholesterol modification of the ER-localized SMO, and SMO cholesterylation accelerated its exit from ER. The GRAMD1/ASTER sterol transport proteins facilitated cholesterol transfer to ER from PM, resulting in increased SMO cholesterylation and enhanced Hh signaling. Collectively, we reveal a regulatory role of GRAMD-mediated cholesterol transport in ER-resident SMO maturation and Hh signaling.

Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion.

High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

Topological characteristics of international business cycle synchronization: A network analysis of the BRI economies.

Based on the GDP constant 2010 US$ from the World Bank, this paper uses the instantaneous quasi-correlation coefficient to measure the business cycle synchronization linkages among 53 Belt and Road Initiative (BRI) economies from 2000 to 2019, and empirically studies the topological characteristics of the Business Cycle Synchronization Network (BCSN) with the help of complex network analysis method. The main conclusions are as follows: First, the BCSN density and efficiency of BRI economies are still low, and it presents a topological feature of "small world". Second, the individual characteristics of the economies in the network are obviously different. Among them, China's relative influence is significantly increased, but its betweenness centrality level is still low. Third, since the inception of BRI, the topological characteristics of BCSN of BRI economies have undergone great changes, and their topological evolution has gradually reflected the characteristic of self-stability.

Cholesterylation of Smoothened is a calcium-accelerated autoreaction involving an intramolecular ester intermediate.

Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous SmoD99E/D99E knockin mice are embryonic lethal with severe developmental delay, demonstrating that cholesterylation of CRD is required for full-length SMO activation. Our work reveals the unique autocatalytic mechanism of SMO cholesterylation and an unprecedented role of calcium in Hh signaling.

The 3-beta-hydroxysteroid-Delta(8), Delta(7)-isomerase EBP inhibits cholesterylation of Smoothened.

Hedgehog (Hh) pathway plays a central role in vertebrate embryonic development and carcinogenesis. The G-protein coupled receptor-like protein Smoothened (SMO) is one of the major members in Hh pathway. Covalent modification of cholesterol on the 95th asparagine (D95) of human SMO, which is regulated by Hh and PTCH1, is critical for SMO activation. However, it is not known whether SMO cholesterylation is regulated by other proteins. In this study, we identified Emopamil binding protein (EBP, also known as 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase) as a SMO-interacting protein. Overexpression of EBP suppressed SMO cholesterylation and Hh pathway activity, whereas genetic disruption of EBP enhanced SMO cholesterylation and the downstream signaling. EBP-mediated inhibition of SMO cholesterylation was independent of its isomerase activity, but dependent on the C-terminus of EBP that was required for SMO binding. The X-linked dominant chondrodysplasia punctate 2 (CDPX2)-associated EBP mutants inhibited SMO cholesterylation too. Together, this study shows that EBP modulates SMO cholesterylation through direct binding and suggests a possible mechanism of CDPX2 pathogenesis.

Quantile regression models for survival data with missing censoring indicators.

The quantile regression model has increasingly become a useful approach for analyzing survival data due to its easy interpretation and flexibility in exploring the dynamic relationship between a time-to-event outcome and the covariates. In this paper, we consider the quantile regression model for survival data with missing censoring indicators. Based on the augmented inverse probability weighting technique, two weighted estimating equations are developed and corresponding easily implemented algorithms are suggested to solve the estimating equations. Asymptotic properties of the resultant estimators and the resampling-based inference procedures are established. Finally, the finite sample performances of the proposed approaches are investigated in simulation studies and a real data application.

A Smooth Nonparametric Approach to Determining Cut-Points of A Continuous Scale.

The problem of determining cut-points of a continuous scale according to an establish categorical scale is often encountered in practice for the purposes such as making diagnosis or treatment recommendation, determining study eligibility, or facilitating interpretations. A general analytic framework was recently proposed for assessing optimal cut-points defined based on some pre-specified criteria. However, the implementation of the existing nonparametric estimators under this framework and the associated inferences can be computationally intensive when more than a few cut-points need to be determined. To address this important issue, a smoothing-based modification of the current method is proposed and is found to substantially improve the computational speed as well as the asymptotic convergence rate. Moreover, a plug-in type variance estimation procedure is developed to further facilitate the computation. Extensive simulation studies confirm the theoretical results and demonstrate the computational benefits of the proposed method. The practical utility of the new approach is illustrated by an application to a mental health study.

Smoothed Rank Regression for the Accelerated Failure Time Competing Risks Model with Missing Cause of Failure.

This paper examines the accelerated failure time competing risks model with missing cause of failure using the monotone class rank-based estimating equations approach. We handle the non-smoothness of the rank-based estimating equations using a kernel smoothed estimation method, and estimate the unknown selection probability and the conditional expectation by non-parametric techniques. Under this setup, we propose three methods for estimating the unknown regression parameters based on 1) inverse probability weighting, 2) estimating equations imputation and 3) augmented inverse probability weighting. We also obtain the associated asymptotic theories of the proposed estimators and investigate the estimators' small sample behaviour in a simulation study. A direct plug-in method is suggested for estimating the asymptotic variances of the proposed estimators. A real data application based on a HIV vaccine efficacy trial study is considered.

Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Copper-mediated C(sp(3))-H azidation with Me3SiN3: synthesis of imidazoles from ketones and aldehydes.

The efficient construction of 2,4,5-trisubstituted imidazoles, through a copper-mediated three-component reaction involving ketones, aldehydes, and Me3SiN3, has been developed. During the process, 4 C-N bonds were formed sequentially. Experimental results and DFT calculations suggested that azidation of the alpha methylene group of the ketone was the key C-N bond-forming step.

Mechanism of the rhodium(iii)-catalyzed alkenylation reaction of N-phenoxyacetamide with styrene or N-tosylhydrazone: a computational study.

A systematic density functional theory study has been conducted to examine the mechanisms involved in the rhodium(iii)-catalyzed alkenylation of N-phenoxyacetamide with two different substrates (i.e., styrene and N-tosylhydrazone). The density functional theory calculations indicated that the reaction of the N-tosylhydrazone substrate resulted in the formation of a Rh(v)-nitrene intermediate via the cleavage of the O-N bond of N-phenoxyacetamide, whereas the styrene substrate resulted in an Rh(i) species through consecutive ß-H elimination and H migration steps to the internal oxidant. The differences observed between the N-tosylhydrazone and styrene systems were attributed to differences in the reactivity of their Rh(v)-nitrene intermediates. For example, the N-tosylhydrazone formed a five-membered Rh(v)-nitrene intermediate, which was readily reduced to a Rh(iii) species by tautomerization, whereas this pathway was energetically unfavorable for the styrene substrate.

DFT Studies on the Mechanism of the Rhodium(III)-Catalyzed C-H Activation of N-Phenoxyacetamide.

A density functional theory (DFT) study has been conducted to elucidate the mechanism of the rhodium(III)-catalyzed C-H activation of N-phenoxyacetamide, where the amido component of an internal oxidant serves as a leaving group. The impact of different substrates (alkynes versus cyclopropenes) on the reaction mechanism has been discussed in detail. The pathway for cyclopropene substrate proceeded via a Rh(V) nitrene, while Rh(III) remained unchanged throughout the pathway for alkyne substrate. The C-O bond-forming reductive elimination and O-N bond cleavage steps simultaneously occurred for the alkyne substrate. However, the C-O bond was formed by an electrocyclization from a Rh(III) intermediate for the cyclopropene substrate. The energy profiles for the cyclopropene substrate were accompanied by a change in spin-state because the triplet spin state of a Rh(V) nitrene complex is lower than that of the singlet spin state.

A mechanistic study of Pd(OAc)2-catalyzed intramolecular C-H functionalization reaction involving CO/isonitrile insertion.

The mechanism of the Pd(OAc)2-catalyzed intramolecular C-H functionalization reaction involving CO/isonitrile insertion was investigated with the aid of density functional theory calculations at the B3LYP level. The similarity between the CO and isonitrile systems includes the following: (1) the anagostic bonding mechanism rather than the concerted metallation-deprotonation (CMD) mechanism is operative for the C-H cleavage step, (2) the CO/isonitrile insertion step is rate-determining, and (3) the C-H activation and CO/isonitrile insertion steps are accomplished with Pd(II) and Pd(III), respectively. For the reaction including isonitrile insertion, the arene C-H activation step occurs after deprotonation of the imino group. However, for reaction including CO insertion, the arene C-H activation step is the first step of the reaction mechanism. The difference between CO and isonitrile systems in the reaction mechanism can be attributed to the difference in the oxidants used. In the reaction including isonitrile insertion, the high endergonicity of the oxidation step suppresses prior C-H activation and favors prior deprotonation of the imino group. In the reactions including CO insertion, the low endergonicity of the oxidation step allows prior C-H activation to occur.

Rhodium(III)-catalyzed C-H/C-C activation sequence: vinylcyclopropanes as versatile synthons in direct C-H allylation reactions.

Succession of C-H activation and C-C activation was achieved by using a single rhodium(III) catalyst. Vinylcyclopropanes were used as versatile coupling partners. Mechanistic studies suggest that the olefin insertion step is rate-determining and a facile ß-carbon elimination is involved, which represents a novel ring opening mode of vinylcyclopropanes.