Identification of two critical amino acid residues in short-chain aldehyde-responsive odorant receptors.

Mammalian odorant receptors (ORs) are crucial for detecting a broad spectrum of odorants, yet their functional expression poses a significant challenge, often requiring Receptor-transporting proteins (RTPs). This study examines mouse Olfr733 and Olfr732, which, despite high homology, show different functional expression profiles in heterologous cell systems. Our research aimed to identify key amino acids impacting Olfr733's functional expression. We discovered that G112FBW3.40 and L148PBW4.49 (Ballesteros-Weinstein numbering in superscript) substitutions in Olfr732 markedly enhance its RTP-independent expression and ligand responsiveness, mirroring Olfr733. These substitutions, particularly Phe112 and Leu148, are crucial for aldehyde recognition and membrane localization in Olfr733, respectively. While Olfr732-type ORs are conserved across species, Olfr733-types, unique to specific rodents, appear to have evolved from Olfr732, with Pro148 enhancing membrane expression and aldehyde sensitivity. Mouse ORs with ProBW4.49 tend to exhibit improved membrane expression compared to their paralogs, especially when co-expressed with RTP1S. This study concludes that the Pro residue in the fourth transmembrane domain significantly contributes to the structural stability of certain olfactory receptors, highlighting the intricate molecular mechanisms underlying OR functionality and evolution.

Identification and Characterization of Proteins That Are Involved in RTP1S-Dependent Transport of Olfactory Receptors.

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

Antagonistic interactions between odorants alter human odor perception.

The olfactory system uses hundreds of odorant receptors (ORs), the largest group of the G-protein-coupled receptor (GPCR) superfamily, to detect a vast array of odorants. Each OR is activated by specific odorous ligands, and like other GPCRs, antagonism can block activation of ORs. Recent studies suggest that odorant antagonisms in mixtures influence olfactory neuron activities, but it is unclear how this affects perception of odor mixtures. In this study, we identified a set of human ORs activated by methanethiol and hydrogen sulfide, two potent volatile sulfur malodors, through large-scale heterologous expression. Screening odorants that block OR activation in heterologous cells identified a set of antagonists, including ß-ionone. Sensory evaluation in humans revealed that ß-ionone reduced the odor intensity and unpleasantness of methanethiol. Additionally, suppression was not observed when methanethiol and ß-ionone were introduced simultaneously to different nostrils. Our study supports the hypothesis that odor sensation is altered through antagonistic interactions at the OR level.

Development of an odorant sensor with a cell-free synthesized olfactory receptor and a graphene field-effect transistor.

Animals sense odorants using olfactory receptors. Many trials have been conducted to develop artificial odorant sensors using olfactory receptors. However, the development has been hindered by the difficulty in obtaining olfactory receptors. In this study, we expressed an olfactory receptor, cOR52, using a wheat germ cell-free synthesis system. The functionality of the expressed cOR52 was confirmed by ligand concentration-dependent interactions with the mini-G protein. The expressed cOR52 was immobilized on a graphene field-effect transistor. The cOR52-modified graphene field-effect transistor exhibited a ligand-specific response between 100 nM and 100 µM. This approach seems to be applicable for other olfactory receptors. Therefore, it will be possible to develop an odorant sensor equipped with various olfactory receptors by this method.

Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors.

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.

Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors.

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.

Axonal Odorant Receptors Mediate Axon Targeting.

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.

The N-terminal region of RTP1S plays important roles in dimer formation and odorant receptor-trafficking.

Receptor-transporting protein 1S (RTP1S) is an accessory protein that mediates the transport of mammalian odorant receptors (ORs) into the plasma membrane. Although most ORs fail to localize to the cell surface when expressed alone in nonolfactory cells, functional expression of ORs is achieved with the coexpression of RTP1S. However, the mechanism for RTP1S-mediated OR trafficking remains unclear. In this study, we attempted to reveal the mode of action and critical residues of RTP1S in OR trafficking. Experiments using N-terminal truncation and Ala substitution mutants of RTP1S demonstrated that four N-terminal amino acids have essential roles in OR trafficking. Additionally, using recombinant proteins and split luciferase assays in mammalian cells, we provided evidence for the dimer formation of RTP1S. Furthermore, we determined that the 2nd Cys residue is required for the efficient dimerization of RTP1S. Altogether, these findings provide insights into the mechanism for plasma membrane transport of ORs by RTP1S.

Anticancer saponin OSW-1 is a novel class of selective Golgi stress inducer.

OSW-1 is a plant-derived natural product proposed to selectively kill cancer cells by binding to members of the oxysterol binding protein family, thereby disrupting lipid/sterol homeostasis. However, how these protein-ligand interactions mediate cell death signaling has remained elusive. Here, we discovered that OSW-1 selectively activates the Golgi stress response leading to apoptosis, providing a mechanistic basis for the anticancer activity of OSW-1.

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to "de-orphanize" these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Vapor detection and discrimination with a panel of odorant receptors.

Olfactory systems have evolved the extraordinary capability to detect and discriminate volatile odorous molecules (odorants) in the environment. Fundamentally, this process relies on the interaction of odorants and their cognate olfactory receptors (ORs) encoded in the genome. Here, we conducted a cell-based screen using over 800 mouse ORs against seven odorants, resulting in the identification of a set of high-affinity and/or broadly-tuned ORs. We then test whether heterologously expressed ORs respond to odors presented in vapor phase by individually expressing 31 ORs to measure cAMP responses against vapor phase odor stimulation. Comparison of response profiles demonstrates this platform is capable of discriminating between structural analogs. Lastly, co-expression of carboxyl esterase Ces1d expressed in olfactory mucosa resulted in marked changes in activation of specific odorant-OR combinations. Altogether, these results establish a cell-based volatile odor detection and discrimination platform and form the basis for an OR-based volatile odor sensor.

Modification of the response of olfactory receptors to acetophenone by CYP1a2.

Olfaction is mediated by the binding of odorant molecules to olfactory receptors (ORs). There are numerous proteins in the nasal mucus, and they contribute to olfaction through various mechanisms. Cytochrome P450 (CYP) family members are known to be present in the olfactory epithelium and are thought to affect olfaction by enzymatic conversion of odorant molecules. In this study, we examined the effects of CYPs on the ligand responses of ORs in heterologous cells. Among the CYPs tested, co-expression of CYP1a2 significantly affected the responses of various ORs, including MOR161-2, to acetophenone. Conversion of acetophenone to methyl salicylate was observed in the medium of CYP1a2-expressing cells. MOR161-2-expressing cells exhibited significantly greater responses to methyl salicylate than to acetophenone. Finally, we analyzed the responses of olfactory neurons expressing MOR161-2 in vivo using the phosphorylated ribosomal protein S6 as a marker. MOR161-2 responded to both acetophenone and methyl salicylate in vivo. When the olfactory mucus was washed out by the injection of PBS to mouse nasal cavity, the response of MOR161-2 to acetophenone was reduced, while that to methyl salicylate did not change. Our data suggest that CYP1a2 affects OR activation by converting acetophenone to methyl salicylate.

Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and ß-arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. © 2017 Wiley Periodicals, Inc.

Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory proteins.

Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors.

Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771.

The encapsulin nanocompartment from Rhodococcus erythropolis N771 (Reencapsulin) was expressed and purified in wild-type and C-terminally His-tagged forms. Negative-stained transmission electron microscopy, field-flow fractionation combined with multi-angle light scattering and dynamic light scattering analyses showed that 60 Reencapsulin monomers were assembled as a spherical particle with a diameter of 28 nm. Heterogeneous guest proteins such as EGFP and firefly luciferase were packaged into the internal cavity of the Reencapsulin nanocompartment by fusing the C-terminal 37-amino-acid sequence of the R. erythropolis N771 DypB peroxidase to the C-terminus. Reencapsulin has the potential to package target proteins in its internal cavity and/or display them on its external surface, making it a feasible carrier for nanotechnology applications.

An improved bioluminescence-based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor.

The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein α-subunit (Gpa1p) with the olfactory-specific Gα(olf), the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast.

The N-terminal replacement of an olfactory receptor for the development of a yeast-based biomimetic odor sensor.

For the development of a biomimetic odor-sensing system, we investigated the effects of replacing the N-terminus of an olfactory receptor (OR) on its functional expression in the budding yeast, Saccharomyces cerevisiae. Using the mouse olfactory receptor OR226 (mOR226), three types of chimeric ORs were constructed by replacing N-terminal regions of mOR226 with the corresponding regions of the rat I7 receptor, which is known to be functionally expressed in yeast. The replacement of the N-terminal region of mOR226 dramatically affected the expression and localization of the receptor and improved the sensing ability of the yeast cells for the odorant. Furthermore, the replacement of the endogenous yeast G-protein α subunit (Gpa1) by the OR-specific G(olf) drastically elevated the odorant-sensing ability of the yeast cells and caused the cells to display a dose-dependent responsiveness to the odorant. Because of the suitability of yeast cells for screening large-scale libraries, the strategy presented here would be useful for the establishment of advanced biomimetic odor-sensing systems.