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2025-01-15 Update From: SLTechnology News&Howtos shulou NAV: SLTechnology News&Howtos > IT Information >
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This article comes from the official account of Wechat: ID:fanpu2019, author: Chen Qingchao
The sense of smell is one of the earliest senses formed in the human body, and its importance may be ignored because it is too common in our lives. The sense of smell not only plays a role in enjoying good food and feeling the danger of the environment, it is also closely related to memory and emotion. So, why can we smell it? This is a very basic but extremely complex problem. The exploration of olfactory receptors is the key to finding the answer.
Author | Chen Qingchao (postdoctoral fellow, MRC Molecular Biology Laboratory, University of Cambridge)
In the diversified material world, there is a world that we can't see or touch, but we can really feel it. It comes either from the fragrance of the soil and grass after the rain, or from the temptation of delicious food on the table, it even exists in the memory, connected with the trickle of emotion, this is the "world of smell".
There are millions of different kinds of odors, each of which is made up of hundreds of chemical molecules with different properties. Why can we feel and distinguish such complex and diverse smells? For a long time, this is one of the most important scientific problems that are seldom explored in biology.
Figure 1. Odor molecules contained in the odors of common fruits and vegetables (strawberries, tomatoes, and blueberries). Each circle and square represents an odor molecule. | Source: salk.edu in fact, "perception" and "discrimination" are two different biological problems: one is how our olfactory system perceives complex and diverse odor molecules, and the other is how our nervous system decodes odor signals to form different olfactory perception. This paper mainly focuses on the first problem and shares with you the exploration of the structure of olfactory receptors in the past decades.
Searching for olfactory receptors is one of the earliest senses formed in the human body, which is a very complex sensory response. Through millions of olfactory nerves, we are able to sense and distinguish a variety of small molecular compounds with different structural properties, that is, odor molecules, even at very low concentrations (micromoles or even nanomoles). [2]
The nasal mucosa of the human body is covered with tissue called olfactory epithelium, in which a large number of olfactory sensory neurons grow and connect with each other. Olfactory sensory nerve cells extend through cilia to the mucous layer in the nasal cavity. The process by which we smell a certain smell is as follows (figure 1): odor molecules enter the nasal mucosa and are sensed by the primary cilia of olfactory sensory neurons to activate olfactory nerve cells and produce chemical signals; these chemical signals trigger nerve cells to generate electrical signals, which are then transmitted through the olfactory nerve to the olfactory bulb and then to the olfactory cortex (the cortical area of the brain responsible for olfactory processing). In the olfactory layer, the brain analyzes and recognizes the incoming olfactory information. Finally, the processing of olfactory nerve signals forms semantic representations that describe various odors, such as coffee, rose, mango, and so on.
Figure 2. A schematic diagram of the human olfactory system. From odor perception, signal transmission to final information processing. | Image source: nobelprize.org for a long time, one of the key issues in the field of olfactory research is how cells sense complex and diverse odor molecules. A reasonable assumption is that there is a special protein called olfactory (odor) receptor (Ordorant Receptor,OR) on olfactory sensory nerve cells, which is used to detect odor molecules. For a long time, scientists have been trying to find these special olfactory receptor proteins.
In the mid-1980s, a series of physiological and biochemical experiments conducted by different research groups showed that odor activated olfactory sensory neurons were mediated by G protein dependent pathways.
G protein is a very important kind of signal transduction molecules in cells. It works with G protein coupled receptor (GPCR) to transmit signals produced by various signal factors such as hormones and neurotransmitters to cells, and further regulates the functions of enzymes, ion channels, transporters and other proteins. In olfactory neurons, G protein mediates the activation of adenylate cyclase, the increase of intracellular cyclic adenosine monophosphate (cAMP) concentration, the activation of cAMP gated ion channels and neuronal depolarization.
At the same time, some olfactory specific genes have been cloned, including genes encoding G protein and cAMP gated ion channels, which further confirmed the important role of G protein signal pathway in odor signal transduction. These studies strongly suggest that olfactory receptors are likely to be G protein coupled receptors (GPCR).
In 1991, Linda Buck and Richard Axel published a groundbreaking study in the journal Science that cloned and identified the olfactory receptor GPCR gene family from rats for the first time [6]. Through further analysis, they also proved that these receptors are only expressed in rat olfactory epithelial cells, not in the other eight tissues, including brain, retina and liver. In addition, in order to estimate the size of the olfactory gene family, they further used a mixture of DNA as probes to screen rat genomic libraries. The results of screening at that time showed that the haploid genome of rats contained at least 50-1000 olfactory receptor genes.
Buck and Axel then worked independently, further discovered the existence of olfactory receptor GPCR gene in human olfactory tissue, and confirmed their important role in the human olfactory system.
These pioneering work laid an important foundation for us to understand and study the mysterious sense of smell, and the two men won the 2004 Nobel Prize in Physiology or Medicine.
Figure 3. The 2004 Nobel Prize in Physiology or Medicine was jointly awarded to Richard Axel (left) and Linda B. Buck (right) for their "discovery of odor receptors and olfactory system structures". | Picture Source: nobelprize.org2004 years later, the completion of the Human Genome Project made it possible to identify and classify human olfactory receptor genes, which further promoted the development of olfactory receptor research.
Now, we know that olfactory receptors are mainly G-protein coupled receptors (GPCR) with seven transmembrane structures. GPCR has more than 800 family members in the human body and is the largest family of cell surface receptors in eukaryotes. They are involved in the regulation of almost all human life activities. Because of this, GPCR has become the "star molecule" of scientific research and an important target of drug development. About 1/3 of all drugs approved by the U.S. Food and Drug Administration (FDA) work by targeting the activity of different GPCR [7]. Of all the GPCR in the human body, about 400 members are classified as olfactory receptors, accounting for half of the GPCR members, which is the largest protein family.
The dilemma of structural analysis of olfactory receptors since the first discovery of olfactory receptors in 1991, structural biologists have been trying to analyze the structure of olfactory receptors in order to clarify the mechanism by which olfactory receptors recognize odor molecules. However, in the past 30 years, the analysis of olfactory receptor structure has not progressed smoothly and is faced with many challenges.
First of all, most of the human olfactory receptors are mainly expressed in nasal nerve cells, and the expression level is low. Therefore, it is difficult to obtain a sufficient amount of protein (usually at the milligram level) from human tissue samples for structural analysis. The effect of heterologous expression (expressed in animal cells or bacteria) is not ideal, not only the level of expression is very low, but also can not have biological activity due to misfolding.
Second, in order to analyze the protein structure of GPCR, we need to bind to some specific high affinity ligand molecules, that is, suitable odor molecules. However, due to the huge chemical diversity of odor molecules and the large number of olfactory receptors, there is still a lack of an efficient method to determine which odor molecules a given olfactory receptor interacts with.
Now academia has gradually realized that each olfactory receptor can interact with a subset of all potential odor molecules, one odor molecule can activate multiple olfactory receptors, and different receptors have different affinity for different odor molecules. Due to the complexity of this interaction, a large number of olfactory receptors do not find suitable ligands for odor molecules, and these receptors are called "orphan receptors" (orphan receptors). At present, a lot of "orphan" research work is under way to develop effective screening methods to find suitable ligands for orphan receptors. In addition, most of the volatile odor molecules are hydrophobic and their solubility is very low, which greatly increases the difficulty of the preparation of odor molecular ligands.
Third, as an important molecule for signal sensing and transduction on the cell membrane, GPCR is a highly dynamic protein molecule, which is constantly changing in various conformations such as inactivation, semi-activation, activation and coupling with different regulatory molecules. Therefore, like most other GPCR, a difficulty in olfactory receptor purification is to stabilize the receptor protein in a specific conformation, which is very important for the formation of protein crystals.
In recent years, several research groups have developed a number of methods to stabilize the different conformations of GPCR, including, but not limited to, obtaining stable receptor mutants for protein crystallization by stability mutagenesis; stabilizing the fully active structure of GPCR coupled with G protein by binding "mini G protein (miniGs)"; binding to high affinity small molecular ligands (including agonists, antagonists, reverse agonists, etc.) A new type of nano-antibody (Nanobody) was developed to stabilize the conformation of different GPCR complexes. For a particular GPCR, many different methods need to be tried to stabilize a specific conformation, which is a very time-consuming and laborious process.
Dawn: from insects to humans, structural biology has stepped into the era of frozen electron microscopy from crystal diffraction. In a complete single particle freeze electron microscope technique, the purified protein is instantly frozen in a thin layer of amorphous vitreous ice and then imaged by transmission electron microscope. hundreds of thousands to millions of protein particle data were recorded-for 3D reconstruction and accurate modeling (figure 4). Compared with traditional crystallographic methods, single particle freeze electron microscopy (Cryo-EM) has obvious advantages in analyzing the high resolution structure of biological macromolecules, such as no need to obtain crystals, small amount of samples and various methods of sample preparation, and has been widely used to analyze the complex structure of GPCR and downstream proteins, which brings dawn for the analysis of olfactory receptor structure.
Figure 4. The basic work flow of single particle freezing electron microscope (Single Particle Cryo-EM): the purified protein sample is placed on the grid, then vitrified with liquid ethane, and the protein particles embedded in the thin ice will have various random directions, which will be imaged by transmission electron microscope (TEM), and then reconstructed through a series of image processing. Finally, the high-resolution protein freeze electron microscope structure can be obtained. | Image Source: in pdf.medrang.co.kr2018, researchers at the Ruta Laboratory of Rockefeller University in the United States analyzed the single particle freeze electron microscopic structure of the odor coreceptor Orco of a parasitic wasp [9]. Unlike mammals, insect odor receptors are not GPCR, but gated ion channels, which are heteropolymer ion channels composed of odor receptor OR and highly conserved coreceptor Orco. This ion channel is like a hole through which charged particles flow, opening only when the receptor encounters its target odor molecule, activating olfactory sensory cells. For a long time, there has been controversy in the scientific community whether Orco can function as an independent olfactory receptor, and there is no unified model of insect odor perception and signal transduction. This work shows for the first time the fine structure of the homologous tetramer of insect odor coreceptor Orco, which provides conclusive evidence for determining that "insect olfactory coreceptor Orco can form a new class of heteropoly ligand gated ion channels". The structure is analyzed and its function is confirmed, which provides an important new insight for understanding the olfactory mechanism around insects.
In 2021, another study, also from the Ruta laboratory, analyzed the freeze electron microscopic structure of the olfactory receptor OR5 of a terrestrial insect jumping its bristle tail [10] (figure 5). By comparing the structures of three different odor molecules bound by OR5, the researchers found that the binding of odor molecules mainly depends on hydrophobic interactions and lacks the strict geometric constraints inherent in other intermolecular forces that often mediate ligand recognition, such as hydrogen bonds.
Hydrophobic interaction is a force that stabilizes the three-dimensional structure of proteins, which usually occurs in two or more non-polar amino acid residues. When they are in a polar environment (the most common is water), their "aversion" to water causes them to get close to each other in some way in order to interact with the polar environment as little as possible. This non-specific weak interaction provides a new mechanism for explaining why an olfactory receptor can recognize different odors, which is different from the classical lock-and-key model of many other receptor ligands. However, the non-specificity of OR5 receptor does not mean that it has no preference. Although it can bind to many different odor molecules, it is not sensitive to many other odor molecules. In addition, if you make a simple mutation of some amino acids in the binding pocket, that is, re-change the receptor, the receptor can bind to molecules you don't like. The discovery also helps explain why insects have been able to mutate millions of odor receptors to adapt to the various environments they encounter and form a unique way of life.
Figure 5. Frozen electron microscopic structure of olfactory receptor OR5 from bristle and tail of terrestrial insects. When the odor molecule binds to the olfactory receptor, the channel pore of the olfactory receptor expands (pink). | Source: rockefeller.edu 's structural biology studies on insect olfactory receptors have brought us a lot of new understanding of the odor recognition mechanism, but humans and insects are different after all. We urgently need the high-resolution structure of human olfactory receptors to unveil the "veil" of human olfactory perception.
Until March 2023, an article published in the journal Nature revealed the mystery of the structure of human olfactory receptors for the first time [11].
In this work, the researchers chose an olfactory receptor called OR5E2. They chose this receptor because it is expressed not only in olfactory nerve cells, but also in other non-olfactory organs such as the prostate, suggesting that it is more likely to be expressed in heterologous systems. In other words, it is easier to get enough protein.
Matching molecules for this receptor are also easy to obtain. Previous studies have shown that this receptor can bind and respond to the water-soluble short-chain fatty acid (short chain fatty acids, SCFAs) odor molecule-propionic acid. Short-chain fatty acids are a kind of signal molecules produced by intestinal flora, which are easy to volatilize, have special irritating odor, and play an important role in the occurrence and development of many diseases.
In addition, OR5E2 are more conservative in evolution, probably because they recognize odors that are vital to the survival of animals in many species, and the researchers speculate that this olfactory receptor may be more evolutionarily constrained by stability.
In short, through these strategies, the researchers skillfully avoided the challenges of low expression levels of most olfactory receptors, low solubility of most volatile odors and high instability of purified olfactory receptors. Through fusion expression of mini G protein and binding of G β 1 γ 2 protein and nano-antibody Nb35, the researchers stabilized an activated state of OR5E2 binding to propionic acid, and analyzed its three-dimensional high-resolution structure by freeze electron microscopy (figure 6).
Figure 6. The 3D structure of human odor receptor OR51E2 (green). Purple, red and blue spirals and entanglements are receptor-coupled G protein subunits, and orange is a nanoantibody used to stabilize the structure. | Source: Kristina Armitage / Quanta Magazine; Sources: NIH / NIDCD; ArtBalitskiy / iStock; Alhontess / iStock in this structure, the OR51E2 receptor locks the odor molecule propionic acid in a small closed binding pocket. In this small pocket, propionic acid binds to OR51E2 through two types of interactions: polar interactions (hydrogen bonds and ion bonds), and non-specific hydrophobic interactions. Therefore, the way in which OR51E2 binds odor molecules is different from that of insect odor-gated ion channels, which seems to be more selective.
Many olfactory receptors can respond to a variety of odorants with different chemical properties, while OR51E2 seems to bind only to short-chain fatty acids. So what factors determine this selectivity? Further analysis of this structure shows that the selectivity of OR51E2 for short-chain fatty acids stems from the volume of the closed binding pocket (31), which can hold short-chain fatty acids, such as acetic acid and propionic acid, but prevents longer fatty acid chains from binding. Therefore, the researchers believe that the volume of the binding pocket is an important selective factor for odor molecules.
As the first published activated structure of the ligand binding of human olfactory receptors and odor molecules, this is a gratifying research result, which gives us a visual view of how odor molecules bind to olfactory receptors for the first time, although it is not perfect in many aspects, such as the coupling of receptors and G proteins.
The binding of ligands to GPCR usually causes conformational changes, which leads to G-protein coupling and further transmits the signal to G-protein. Under physiological conditions, mammalian olfactory receptors can bind to two highly homologous G proteins G α olf and G α s. In this structure, instead of coupling G α olf or G α s, the researchers used fusion expression of miniG α s and binding of G β 1 γ 2 and nano-antibody Nb35 to stabilize the structure of receptor and G protein heterotrimer. Although some interactions between olfactory receptors and G proteins have been found, this is not enough to explain the interaction mechanism with the real G α olf and G α s in vivo.
On May 24, 2023, Sun Jinpeng Laboratory of the School of basic Medicine of Shandong University published a work online in the journal Nature. The mouse trace amine olfactory receptor TAAR9 (mTAAR9) recognized four endogenous amine ligands (phenylethylamine, dimethylcyclohexylamine, cadaverine, spermidine) and coupled with downstream G α s and G α olf proteins [12].
Trace amine-associated receptors (trace amine-associated receptor,TAAR) are evolutionarily conserved G-protein-coupled receptors in vertebrates, which can sense trace amines (trace amine) at nanomole concentration. Trace amines are formed by decarboxylation of amino acids. for animals, they can be used as odor molecules that sense a series of stimuli, such as judging the existence of predators or prey, the proximity of mating partners and the deterioration of food. and according to the smell to cause intraspecific or interspecific attraction or aversion response. In recent years, more and more studies have shown that trace amines in human body are associated with a variety of mental disorders, so TAAR has become a potential new target for the treatment of mental diseases such as schizophrenia, depression and drug addiction.
Figure 7. The structure of mouse olfactory receptor mTAAR9, Gas and Gaolf protein trimer complexes bound by different ligands. | Source: Nature in this study, researchers found that the olfactory receptor TAAR forms a pair of disulfide bonds between the N-terminal and the second extracellular segment, which has never been found in other known GPCR receptors, and these disulfide bonds are essential for mTAAR9 to recognize ligands and stabilize the extracellular conformation of the receptor.
A single TAAR olfactory receptor can recognize a variety of amine odor molecules, and the same amine odor molecule can also be recognized by multiple olfactory receptors. The complex nature of this interaction is an important basis for olfactory sensory amine molecules. This study found a general structural motif for mTAAR9 to recognize amine odor molecules and a combined structural motif for recognizing different amine odor molecules, which provides a new insight for amine odor molecular recognition.
It is worth noting that the researchers also analyzed the molecular structure of the mTAAR9 receptor coupled with two downstream G proteins G α s and G α olf. As the complex structure of olfactory receptor and G α olf determined in the first experiment, it provides an important understanding for the complete activation of olfactory receptor in mammals after G protein coupling.
With the help of frozen electron microscopy, the structural analysis of olfactory receptors has begun to emerge, and a greater challenge will follow.
The above structure reveals only an active conformation, but in the physiological state, the olfactory receptor is highly dynamic. With the high development of artificial intelligence in the field of protein structure prediction, researchers also try to show the dynamic changes of receptors through computer simulation to improve the theoretical model, but this can not be completely equal to the structural changes in the real physiological state. We need to analyze the structure of more olfactory receptors under different time dynamics, and develop high-resolution dynamic monitoring methods of receptor proteins to help us open the complete biological "black box" of olfactory perception.
In recent years, with the continuous development of sequencing technology, the expression of olfactory receptors has been found in more non-olfactory tissues, including heart, respiratory tract, kidney, liver, lung, skin, brain and so on. The expression of these olfactory receptors in non-olfactory tissues is both universal and specific. Studies have shown that olfactory receptors expressed outside the nasal cavity have specific biological functions in specific tissues [13]. Some studies have found that the dysfunction of olfactory receptors is related to the occurrence and development of diseases such as nervous system diseases and tumors. The analysis of the physiological structure of these receptors in non-olfactory tissues provides a new direction and challenge for the study of olfactory receptor structure, and these olfactory receptors are expected to become important drug targets in the future.
Back to the question at the beginning of this article: why can our olfactory system sense and distinguish such complex and diverse odors? Scientifically, we still do not have a complete answer to this question, and the problem seems to become more complicated as we study more and understand more about the structure of olfactory receptors. How olfactory receptors respond selectively to odor molecules in the air is only part of a larger odor puzzle. Researchers still face a more complex challenge: understanding how the brain converts electrochemical signals transmitted by receptors into odor perception.
We still have a long way to go to understand the mysteries of olfactory perception.
reference
[1] https://pubmed.ncbi.nlm.nih.gov/28424010/
[2] https://academic.oup.com/nar/article/50/D1/D678/6362078
[3] https://www.ingentaconnect.com/content/ben/cn/2019/00000017/00000009/art00010
[4] https://www.ncbi.nlm.nih.gov/books/NBK55985/
[5] https://www.science.org/doi/10.1126/
[6] https://pubmed.ncbi.nlm.nih.gov/1840504/
[7] https://www.nature.com/articles/nrd.2017.178
[8] https://zh.wikipedia.org/wiki/%E5%AD%A4%E5%84%BF%E5%8F%97%E4%BD%93
[9] https://www.nature.com/articles/s41586-018-0420-8
[10] https://www.nature.com/articles/s41586-021-03794-8
[11] https://www.nature.com/articles/s41586-023-05798-y
[12] https://www.nature.com/articles/s41586-023-06106-4
[13] https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055368
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