Profiling of FSHR negative allosteric modulators on LH/CGR reveals biased antagonism with implications in steroidogenesis.

Biased signaling has recently emerged as an interesting means to modulate the function of many G protein-coupled receptors (GPCRs). Previous studies reported two negative allosteric modulators (NAMs) of follicle-stimulating hormone receptor (FSHR), ADX68692 and ADX68693, with differential effects on FSHR-mediated steroidogenesis and ovulation. In this study, we attempted to pharmacologically profile these NAMs on the closely related luteinizing hormone/chorionic gonadotropin hormone receptor (LH/CGR) with regards to its canonical Gs/cAMP pathway as well as to ß-arrestin recruitment in HEK293 cells. The NAMs' effects on cAMP, progesterone and testosterone production were also assessed in murine Leydig tumor cell line (mLTC-1) as well as rat primary Leydig cells. We found that both NAMs strongly antagonized LH/CGR signaling in the different cell models used with ADX68693 being more potent than ADX68692 to inhibit hCG-induced cAMP production in HEK293, mLTC-1 and rat primary Leydig cells as well as ß-arrestin 2 recruitment in HEK293 cells. Interestingly, differential antagonism of the two NAMs on hCG-promoted steroidogenesis in mLTC-1 and rat primary Leydig cells was observed. Indeed, a significant inhibition of testosterone production by the two NAMs was observed in both cell types, whereas progesterone production was only inhibited by ADX68693 in rat primary Leydig cells. In addition, while ADX68693 totally abolished testosterone production, ADX68692 had only a partial effect in both mLTC-1 and rat primary Leydig cells. These observations suggest biased effects of the two NAMs on LH/CGR-dependent pathways controlling steroidogenesis. Interestingly, the pharmacological profiles of the two NAMs with respect to steroidogenesis were found to differ from that previously shown on FSHR. This illustrates the complexity of signaling pathways controlling FSHR- and LH/CGR-mediated steroidogenesis, suggesting differential implication of cAMP and ß-arrestins mediated by FSHR and LH/CGR. Together, our data demonstrate that ADX68692 and ADX68693 are biased NAMs at the LH/CGR in addition to the FSHR. These pharmacological characteristics are important to consider for potential contraceptive and therapeutic applications based on such compounds.

Low reversibility of intracellular cAMP accumulation in mouse Leydig tumor cells (MLTC-1) stimulated by human Luteinizing Hormone (hLH) and Chorionic Gonadotropin (hCG).

In order to study the intracellular cAMP response kinetics of Leydig cells to hormones with LH activity, we used MLTC-1 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be followed using oxiluciferin luminescence produced from catalyzed luciferin oxidation. The potencies of the different LHs and CGs were evaluated using areas under the curves (AUC) of their kinetics over 60 min stimulation. All mammalian LHs and CGs tested were found to stimulate cAMP accumulation in these cells. The reversibility of this stimulation was studied by removing the hormone from the culture medium after 10 min of incubation. The ratios of kinetics AUC after removing or not the hormone were used to evaluate the stimulation reversibility of each hormone. Natural and recombinant hLHs and hCGs were found to exhibit slowly reversible activation compared to pituitary rat, ovine, porcine, camel and equine LHs, serum-derived eCG (PMSG) and recombinant eLH/CGs. Carbohydrate side chains are not involved in this phenomenon since natural and recombinant homologous hormones exhibit the same reversibility rates. It is still unknown whether only one human subunit, α or ß, is responsible for this behaviour or whether it is due to a particular feature of the hLH and hCG quaternary structure.

Assessing Gonadotropin Receptor Function by Resonance Energy Transfer-Based Assays.

Gonadotropin receptors belong to the super family of G protein-coupled receptors and mediate the physiological effects of follicle-stimulating hormone (FSHR) and luteinizing hormone (LHR). Their central role in the control of reproductive function has made them the focus of intensive studies. Upon binding to their cognate hormone, they trigger complex signaling and trafficking mechanisms that are tightly regulated in concentration, time, and space. Classical cellular assays often fail to capture all these dynamics. Here, we describe the use of various bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays to investigate the activation and regulation of FSHR and LHR in real-time, in living cells (i.e., transiently expressed in human embryonic kidney 293 cells). Indeed, the dynamics of hormone-mediated heterotrimeric G protein activation, cyclic adenosine-monophosphate (cAMP) production, calcium release, ß-arrestin 2 recruitment, and receptor internalization/recycling was assessed. Kinetics and dose-response analyses confirmed the expected pharmacological and signaling properties of hFSHR and hLHR but revealed interesting characteristics when considering the two major pathways (cAMP and ß-arrestin 2) of the two receptors assessed by BRET. Indeed, the EC50 values were in picomolar range for cAMP production while nanomolar range was observed for ß-arrestin 2 recruitment as well as receptor internalization. Interestingly, the predicted receptor occupancy indicates that the maximal G protein activation and cAMP response occur at <10% of receptor occupancy whereas >90% of activated receptors is required to achieve full ß-arrestin 2 recruitment and subsequent receptor internalization. The rapid receptor internalization was also followed by a recycling phase. Collectively, our data reveal that ß-arrestin-mediated desensitization, internalization, and the subsequent fast recycling of receptors at the plasma membrane may provide a mechanistic ground to the "spare receptor" paradigm. More generally, the novel tools described here will undoubtedly provide the scientific community investigating gonadotropin receptors with powerful means to decipher their pharmacology and signaling with the prospect of pathophysiological and drug discovery applications.

Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures.

Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHß or anti-oLHß antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin αß heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined.

Biased signalling in follicle stimulating hormone action.

Follicle-stimulating hormone (FSH) plays a crucial role in the control of reproduction by specifically binding to and activating a membrane receptor (FSHR) that belongs to the G protein-coupled receptor (GPCR) family. Similar to all GPCRs, FSHR activation mechanisms have generally been viewed as a two-state process connecting a unique FSH-bound active receptor to the Gs/cAMP pathway. Over the last decade, paralleling the breakthroughs that were made in the GPCR field, our understanding of FSH actions at the molecular level has dramatically changed. There are numerous facts indicating that the active FSHR is connected to a complex signalling network rather than the sole Gs/cAMP pathway. Consistently, the FSHR probably exists in equilibrium between multiple conformers, a subset of them being stabilized upon ligand binding. Importantly, the nature of the stabilized conformers of the receptor directly depends on the chemical structure of the ligand bound. This implies that it is possible to selectively control the intracellular signalling pathways activated by using biased ligands. Such biased ligands can be of different nature: small chemical molecules, glycosylation variants of the hormone or antibody/hormone complexes. Likewise, mutations or polymorphisms affecting the FSHR can also lead to stabilization of preferential conformers, hence to selective modulation of signalling pathways. These emerging notions offer a new conceptual framework that could potentially lead to the development of more specific drugs while also improving the way FSHR mutants/variants are functionally characterized.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener's granulomatosis autoantigen.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 107 M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates.

Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by ß2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and ß2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1)·s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.

Factors acting on Mos1 transposition efficiency.

BACKGROUND: Mariner-like elements (MLEs) are widespread DNA transposons in animal genomes. Although in vitro transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the MLEs transposition in vivo. RESULTS: The transposition of Mos1, first isolated from drosophila mauritiana, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first in vitro experimental demonstration that MITEs of MLE origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates Mos1 transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency. CONCLUSION: The study highlights how experimental conditions can bias interpretation of mariner excision frequency and quality. In vitro, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.