Antibody against apolipoprotein-A1, non-alcoholic fatty liver disease and cardiovascular risk: a translational study.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease increasing cardiovascular disease (CVD) morbidity and mortality. Autoantibodies against apolipoprotein A-1 (AAA-1) are a possible novel CVD risk factor promoting inflammation and disrupting cellular lipid homeostasis, two prominent pathogenic features of NAFLD. We explored the role of AAA-1 in NAFLD and their association with CVD risk. METHODS: HepaRG cells and liver sections from ApoE-/- mice exposed to AAA-1 were used for lipid quantification and conditional protein expression. Randomly selected sera from 312 subjects of the Prevention of Renal and Vascular End-stage Disease (PREVEND) general population cohort were used to measure AAA-1. A Fatty Liver Index (FLI) ≥ 60 and a 10-year Framingham Risk Score (FRS) ≥ 20% were used as proxy of NAFLD and high CVD risk, respectively. RESULTS: In-vitro and mouse models showed that AAA-1 increased triglyceride synthesis leading to steatosis, and promoted inflammation and hepatocyte injury. In the 112 PREVEND participants with FLI ≥ 60, AAA-1 were associated with higher FRS, alkaline phosphatase levels, lower HDL cholesterol and tended to display higher FLI values. Univariate linear and logistic regression analyses (LRA) confirmed significant associations between AAA-1, FLI and FRS ≥ 20%, while in adjusted LRA, FLI was the sole independent predictor of FRS ≥ 20% (OR: 1.05, 95%CI 1.01-1.09, P = 0.003). AAA-1 was not an independent FLI predictor. CONCLUSIONS: AAA-1 induce a NAFLD-compatible phenotype in vitro and in mice. Intricate associations exist between AAA-1, CVD risk and FLI in the general population. Further work is required to refine the role of AAA-1 in NAFLD and to determine if the AAA-1 association with CVD is affected by hepatic steatosis.

Optochemical Control of Therapeutic Agents through Photocatalyzed Isomerization.

A Ru(bpy)3 Cl2 photocatalyst is applied to the rapid trans to cis isomerization of a range of alkene-containing pharmacological agents, including combretastatin A-4 (CA-4), a clinical candidate in oncology, and resveratrol derivatives, switching their configuration from inactive substances to potent cytotoxic agents. Selective in cellulo activation of the CA-4 analog Res-3M is demonstrated, along with its potent cytotoxicity and inhibition of microtubule dynamics.

Dynamic Covalent Michael Acceptors to Penetrate Cells: Thiol-Mediated Uptake with Tetrel-Centered Exchange Cascades, Assisted by Halogen-Bonding Switches.

Chalcogen-centered cascade exchange chemistry is increasingly understood to account for thiol-mediated uptake, that is, the ability of reversibly thiol-reactive agents to penetrate cells. Here, reversible Michael acceptors are shown to enable and inhibit thiol-mediated uptake, including the cytosolic delivery of proteins. Dynamic cyano-cinnamate dimers rival the best chalcogen-centered inhibitors. Patterns generated in inhibition heatmaps reveal contributions from halogen-bonding switches that occur independent from the thyroid transporter MCT8. The uniqueness of these patterns supports that the entry of tetrel-centered exchangers into cells differs from chalcogen-centered systems. These results expand the chemical space of thiol-mediated uptake and support the existence of a universal exchange network to bring matter into cells, abiding to be decoded for drug delivery and drug discovery in the broadest sense.

Chemo- and Regioselective Multiple C(sp2 )-H Insertions of Malonate Metal Carbenes for Late-Stage Functionalizations of Azahelicenes.

Chiral quinacridines react up to four times, step-by-step, with α-diazomalonates under RuII and RhII catalysis. By selecting the catalyst, [CpRu(CH3 CN)3 ][PF6 ] (Cp=cyclopentadienyl) or Rh2 (oct)4 , chemo and regioselective insertions of derived metal carbenes are achieved in favor of mono- or bis-functionalized malonate derivatives, respectively, (r.r.>49 : 1, up to 77 % yield, 12 examples). This multi-introduction of malonate groups is particularly useful to tune optical and chemical properties such as absorption, emission or Brønsted acidity but also cellular bioimaging. Density-functional theory further elucidates the origin of the carbene insertion selectivity and also showcases the importance of conformations in the optical response.

Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice.

Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1-/- mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

Oligonucleotide Phosphorothioates Enter Cells by Thiol-Mediated Uptake.

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non-modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA-approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol-mediated uptake. The transient adaptive network produced by dynamic covalent pseudo-disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.

Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks.

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.

The topology of the lymphotoxin ß receptor that accumulates upon endolysosomal dysfunction dictates the NF-κB signaling outcome.

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.

Automated high-content imaging for cellular uptake, from the Schmuck cation to the latest cyclic oligochalcogenides.

Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose-response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.

Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.

In specialized cell types, lysosome-related organelles support regulated secretory pathways, whereas in nonspecialized cells, lysosomes can undergo fusion with the plasma membrane in response to a transient rise in cytosolic calcium. Recent evidence also indicates that lysosome secretion can be controlled transcriptionally and promote clearance in lysosome storage diseases. In addition, evidence is also accumulating that low concentrations of cyclodextrins reduce the cholesterol-storage phenotype in cells and animals with the cholesterol storage disease Niemann-Pick type C, via an unknown mechanism. Here, we report that cyclodextrin triggers the secretion of the endo/lysosomal content in nonspecialized cells and that this mechanism is responsible for the decreased cholesterol overload in Niemann-Pick type C cells. We also find that the secretion of the endo/lysosome content occurs via a mechanism dependent on the endosomal calcium channel mucolipin-1, as well as FYCO1, the AP1 adaptor, and its partner Gadkin. We conclude that endo-lysosomes in nonspecialized cells can acquire secretory functions elicited by cyclodextrin and that this pathway is responsible for the decrease in cholesterol storage in Niemann-Pick C cells.

Strain-Promoted Thiol-Mediated Cellular Uptake of Giant Substrates: Liposomes and Polymersomes.

Simple cyclic disulfides under high tension mediate the uptake of giant substrates, that is, liposomes and polymersomes with diameters of up to 400 nm, into HeLa Kyoto cells. To place them at the surface of the vesicles, the strained disulfides were attached to the head-group of cationic amphiphiles. Bell-shaped dose response curves revealed self-activation of the strained amphiphiles by self-assembly into microdomains at low concentrations and self-inhibition by micelle formation at high concentrations. Poor colocalization of internalized vesicles with endosomes, lysosomes, and mitochondria indicate substantial release into the cytosol. The increasing activity with disulfide ring tension, inhibition with Ellman's reagent, and inactivity of maleimide and guanidinium controls outline a distinct mode of action that deserves further investigation and is promising for practical applications.

Selective Imaging of Late Endosomes with a pH-Sensitive Diazaoxatriangulene Fluorescent Probe.

Late endosomes are a major trafficking hub in the cell at the crossroads between endocytosis, autophagy, and degradation in lysosomes. Herein is disclosed the first small molecule allowing their selective imaging and monitoring in the form of a diazaoxatriangulene fluorophore, 1a (hexadecyl side chain). The compound is prepared in three steps from a simple carbenium precursor. In nanospheres, this pH-sensitive (pKa = 7.3), photochemically stable dye fluoresces in the red part of visible light (601 and 578 nm, acid and basic forms, respectively) with a quantum yield between 14 and 16% and an excited-state lifetime of 7.7-7.8 ns. Importantly, the protonated form 1a·H(+) provokes a specific staining of late endosome compartments (pH 5.0-5.5) after 5 h of incubation with HeLa cells. Not surprisingly, this late endosome marking depends on the intra-organelle pH, and changing the nature of the lipophilic chain provokes a loss of selectivity. Interestingly, fixation of the fluorophore is readily achieved with paraformaldehyde, giving the possibility to image both live and fixed cells.

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes.

UNLABELLED: Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE: Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics.

A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.

The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.

Automated Microscopy and High Content Screens (Phenotypic Screens) in Academia Labs.

Imaged-based screening has been developing extremely quickly in the past 10 years. Academic institutes quickly realized that the discovery capacity of this technology was huge, allowing the automatic detection and quantification of complex cell phenotypes. Associated with chemical or genetic perturbations, high content screening is the method of choice for a deep system biology analysis. The evolution of high-content screening is mainly due to the recent progress in the development of fast and high quality automated imagers and of a plethora of new very bright fluorescent markers, so that almost any cellular element can be seen and imaged. In this paper we review and summarize the major steps in the development of an image-based screening project.

Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake.

Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade exchangers (CAXs) has been observed by many and is increasingly being used in practice, the molecular basis of TMU is essentially unknown. The objective of this study was to develop a general protocol to decode the dynamic covalent networks that presumably account for TMU. Uptake inhibition patterns obtained from the removal of exchange partners by either protein knockdown or alternative inhibitors are aligned with original patterns generated by CAX transporters and inhibitors and patterns from alternative functions (here cell motility). These inclusive TMU patterns reveal that the four most significant CAXs known today enter cells along three almost orthogonal pathways. Epidithiodiketopiperazines (ETP) exchange preferably with integrins and protein disulfide isomerases (PDIs), benzopolysulfanes (BPS) with different PDIs, presumably PDIA3, and asparagusic acid (AspA), and antisense oligonucleotide phosphorothioates (OPS) exchange with the transferrin receptor and can be activated by the removal of PDIs with their respective inhibitors. These findings provide a solid basis to understand and use TMU to enable and prevent entry into cells.

ScreenSifter: analysis and visualization of RNAi screening data.

BACKGROUND: RNAi screening is a powerful method to study the genetics of intracellular processes in metazoans. Technically, the approach has been largely inspired by techniques and tools developed for compound screening, including those for data analysis. However, by contrast with compounds, RNAi inducing agents can be linked to a large body of gene-centric, publically available data. However, the currently available software applications to analyze RNAi screen data usually lack the ability to visualize associated gene information in an interactive fashion. RESULTS: Here, we present ScreenSifter, an open-source desktop application developed to facilitate storing, statistical analysis and rapid and intuitive biological data mining of RNAi screening datasets. The interface facilitates meta-data acquisition and long-term safe-storage, while the graphical user interface helps the definition of a hit list and the visualization of biological modules among the hits, through Gene Ontology and protein-protein interaction analyses. The application also allows the visualization of screen-to-screen comparisons. CONCLUSIONS: Our software package, ScreenSifter, can accelerate and facilitate screen data analysis and enable discovery by providing unique biological data visualization capabilities.

Inhibition of Cell Motility by Cell-Penetrating Dynamic Covalent Cascade Exchangers: Integrins Participate in Thiol-Mediated Uptake.

Integrins are cell surface proteins responsible for cell motility. Inspired by the rich disulfide exchange chemistry of integrins, we show here the inhibition of cell migration by cascade exchangers (CAXs), which also enable and inhibit cell penetration by thiol-mediated uptake. Fast-moving CAXs such as reversible Michael acceptor dimers, dithiabismepanes, and bioinspired epidithiodiketopiperazines are best, much better than Ellman's reagent. The implication that integrins participate in thiol-mediated uptake is confirmed by reduced uptake in integrin-knockdown cells. Although thiol-mediated uptake is increasingly emerging as a unifying pathway to bring matter into cells, its molecular basis is essentially unknown. These results identify the integrin superfamily as experimentally validated general cellular partners in the dynamic covalent exchange cascades that are likely to account for thiol-mediated uptake. The patterns identified testify to the complexity of the dynamic covalent networks involved. This work also provides chemistry tools to explore cell motility and expands the drug discovery potential of CAXs from antiviral toward antithrombotic and antitumor perspectives.

Cyclic Thiosulfonates for Thiol-Mediated Uptake: Cascade Exchangers, Transporters, Inhibitors.

Thiol-mediated uptake is emerging as a powerful method to penetrate cells. Cyclic oligochalcogenides (COCs) have been identified as privileged scaffolds to enable and inhibit thiol-mediated uptake because they can act as dynamic covalent cascade exchangers, i.e., every exchange produces a new, covalently tethered exchanger. In this study, our focus is on the essentially unexplored COCs of higher oxidation levels. Quantitative characterization of the underlying dynamic covalent exchange cascades reveals that the initial ring opening of cyclic thiosulfonates (CTOs) proceeds at a high speed even at a low pH. The released sulfinates exchange with disulfides in aprotic but much less in protic environments. Hydrophobic domains were thus introduced to direct CTOs into hydrophobic pockets to enhance their reactivity. Equipped with such directing groups, fluorescently labeled CTOs entered the cytosol of living cells more efficiently than the popular asparagusic acid. Added as competitive agents, CTOs inhibit the uptake of various COC transporters and SARS-CoV-2 lentivectors. Orthogonal trends found with different transporters support the existence of multiple cellular partners to account for the diverse expressions of thiol-mediated uptake. Dominant self-inhibition and high activity of dimers imply selective and synergistic exchange in hydrophobic pockets as distinguishing characteristics of thiol-mediated uptake with CTOs. The best CTO dimers with hydrophobic directing groups inhibit the cellular entry of SARS-CoV-2 lentivectors with an IC50 significantly lower than the previous best CTO, below the 10 µM threshold and better than ebselen. Taken together, these results identify CTOs as an intriguing motif for use in cytosolic delivery, as inhibitors of lentivector entry, and for the evolution of dynamic covalent networks in the broadest sense, with reactivity-based selectivity of cascade exchange emerging as a distinguishing characteristic that deserves further attention.

A novel strategy to identify drugs that interfere with endosomal lipids.

Lipids are major components of the cell and, like proteins, exhibit much diversity and are highly regulated. And yet, our knowledge of lipids remains limited primarily because their study is difficult. We will use novel Systems Biology approaches, and in particular high content screening techniques, to investigate the mechanisms that regulate the cellular lipid content and function. Our project is to carry out a small compound screen using lipid imaging techniques to identify conditions that interfere with cellular levels and distribution of cholesterol, lysobisphosphatic acid and phosphoinositol-3-phosphate. This forward chemical genetic screen approach should reveal new molecular tools to investigate the molecular mechanism involved in the regulation of these lipids. The aim is to apply chemical proteomic techniques to identify the molecular target(s) of compounds able to affect the intracellular cholesterol regulation and to assess if these are novel druggable targets. This will be the ideal complementary study to the RNAi screen, currently run in our group, as the effect of the inhibition caused by a small molecule can be rapidly reversed when this is removed. Such a small molecule can be administered to a cell or an animal for a very short time to study the function of the target protein and to look at biological mechanisms in a short time-frame. This project is highly interdisciplinary, and will benefit from the help of the screening core facility, currently developed with the support of the NCCR.