Receptor Orthologs Research Articles

Structure and function of neurohypophysial hormones

Vasopressin (VP) and oxytocin (OXT) are neuropeptides that are synthesized in the hypothalamus and stored in/secreted from the neurohypophysis. Although VP and OXT were initially characterized as osmoregulatory and reproductive hormones, respectively, these peptides exert versatile actions not only in peripheral organs but also in the central nervous system via multiple G protein-coupled receptors. Orthologous peptides and receptors have been identified in various animal phyla, reflecting an ancient origin of this hormone family. The aim of this review is to provide basic information on this hormone family and to propose matters to be addressed in future studies. In the earlier sections of this review, we summarize the historical aspect of VP/OXT research as well as the basic features of hormonal peptides and corresponding receptors. The latter sections describe VP/OXT family peptides and their receptors in nonmammalian species, including invertebrates, to introduce the evolutionary aspect of this hormone family. By integrating knowledge from both general and comparative endocrinology perspectives, we highlight current and future research trends about the VP/OXT system.

Developmental and age-related synapse elimination is mediated by glial Croquemort.

Neurons and glia work together to dynamically regulate neural circuit assembly and maintenance. In this study, we show Drosophila exhibit large-scale synapse formation and elimination as part of normal CNS circuit maturation, and that glia use conserved molecules to regulate these processes. Using a high throughput ELISA-based in vivo screening assay, we identify new glial genes that regulate synapse numbers in Drosophila in vivo, including the scavenger receptor ortholog Croquemort (Crq). Crq acts as an essential regulator of glial-dependent synapse elimination during development, with glial Crq loss leading to excess CNS synapses and progressive seizure susceptibility in adults. Loss of Crq in glia also prevents age-related synaptic loss in the adult brain. This work provides new insights into the cellular and molecular mechanisms that underlie synapse development and maintenance across the lifespan, and identifies glial Crq as a key regulator of these processes.

Evolutionary conservation of receptor compensation for stem cell homeostasis in Solanaceae plants.

Stem cell homeostasis is pivotal for continuous and programmed formation of organs in plants. The precise control of meristem proliferation is mediated by the evolutionarily conserved signaling that encompasses complex interactions among multiple peptide ligands and their receptor-like kinases. Here, we identified compensation mechanisms involving the CLAVATA1 (CLV1) receptor and its paralogs, BARELY ANY MERISTEMs (BAMs), for stem cell proliferation in two Solanaceae species, tomato and groundcherry. Genetic analyses of higher-order mutants deficient in multiple receptor genes, generated via CRISPR-Cas9 genome editing, reveal that tomato SlBAM1 and SlBAM2 compensate for slclv1 mutations. Unlike the compensatory responses between orthologous receptors observed in Arabidopsis, tomato slclv1 mutations do not trigger transcriptional upregulation of four SlBAM genes. The compensation mechanisms within receptors are also conserved in groundcherry, and critical amino acid residues of the receptors associated with the physical interaction with peptide ligands are highly conserved in Solanaceae plants. Our findings demonstrate that the evolutionary conservation of both compensation mechanisms and critical coding sequences between receptor-like kinases provides a strong buffering capacity during stem cell homeostasis in tomato and groundcherry.

Considering Caenorhabditis elegans Aging on a Temporal and Tissue Scale: The Case of Insulin/IGF-1 Signaling.

The aging process is inherently complex, involving multiple mechanisms that interact at different biological scales. The nematode Caenorhabditis elegans is a simple model organism that has played a pivotal role in aging research following the discovery of mutations extending lifespan. Longevity pathways identified in C. elegans were subsequently found to be conserved and regulate lifespan in multiple species. These pathways intersect with fundamental hallmarks of aging that include nutrient sensing, epigenetic alterations, proteostasis loss, and mitochondrial dysfunction. Here we summarize recent data obtained in C. elegans highlighting the importance of studying aging at both the tissue and temporal scale. We then focus on the neuromuscular system to illustrate the kinetics of changes that take place with age. We describe recently developed tools that enabled the dissection of the contribution of the insulin/IGF-1 receptor ortholog DAF-2 to the regulation of worm mobility in specific tissues and at different ages. We also discuss guidelines and potential pitfalls in the use of these new tools. We further highlight the opportunities that they present, especially when combined with recent transcriptomic data, to address and resolve the inherent complexity of aging. Understanding how different aging processes interact within and between tissues at different life stages could ultimately suggest potential intervention points for age-related diseases.

The genome of the blind bee louse fly reveals deep convergences with its social host and illuminates Drosophila origins

Social insects' nests harbor intruders known as inquilines,1 which are usually related to their hosts.2,3 However, distant non-social inquilines may also show convergences with their hosts,4,5 although the underlying genomic changes remain unclear. We analyzed the genome of the wingless and blind bee louse fly Braula coeca, an inquiline kleptoparasite of the western honey bee, Apis mellifera.6,7 Using large phylogenomic data, we confirmed recent accounts that the bee louse fly is a drosophilid8,9 and showed that it had likely evolved from a sap-breeder ancestor associated with honeydew and scale insects' wax. Unlike many parasites, the bee louse fly genome did not show significant erosion or strict reliance on an endosymbiont, likely due to a relatively recent age of inquilinism. However, we observed a horizontal transfer of a transposon and a striking parallel evolution in a set of gene families between the honey bee and the bee louse fly. Convergences included genes potentially involved in metabolism and immunity and the loss of nearly all bitter-tasting gustatory receptors, in agreement with life in a protective nest and a diet of honey, pollen, and beeswax. Vision and odorant receptor genes also exhibited rapid losses. Only genes whose orthologs in the closely related Drosophila melanogaster respond to honey bee pheromone components or floral aroma were retained, whereas the losses included orthologous receptors responsive to the anti-ovarian honey bee queen pheromones. Hence, deep genomic convergences can underlie major phenotypic transitions during the evolution of inquilinism between non-social parasites and their social hosts.

Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei

BackgroundPlasmodium lacks an mRNA export receptor ortholog, such as yeast Mex67. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain, suggesting that NTF2-like domain-containing proteins might be associated with mRNA export in Plasmodium. In this study, the relationship between mRNA export and an NTF2-like domain-containing protein, PBANKA_1019700, was investigated using the ANKA strain of rodent malaria parasite Plasmodium berghei.MethodsThe deletion mutant Δ1019700 was generated by introducing gene-targeting vectors into the P. berghei ANKA genome, and parasite growth and virulence were examined. To investigate whether PBANKA_1019700 is involved in mRNA export, live-cell fluorescence imaging and immunoprecipitation coupled to mass spectrometry (IP-MS) were performed using transgenic parasites expressing fusion proteins (1019700::mCherry).ResultsDeletion of PBANKA_1019700 affected the sexual phase but not the asexual phase of malaria parasites. Live-cell fluorescence imaging showed that PBANKA_1019700 localizes to the cytoplasm. Moreover, IP-MS analysis of 1019700::mCherry indicated that PBANKA_1019700 interacts with ubiquitin-related proteins but not nuclear proteins.ConclusionsPBANKA_1019700 is a noncanonical NTF2-like superfamily protein.

Odorant receptor orthologues from moths display conserved responses to cis-jasmone.

In insects, the odorant receptor (OR) multigene family evolves by the birth-and-death evolutionary model, according to which the OR repertoire of each species has undergone specific gene gains and losses depending on their chemical environment, resulting in taxon-specific OR lineage radiations with different sizes in the phylogenetic trees. Despite the general divergence in the gene family across different insect orders, the ORs in moths seem to be genetically conserved across species, clustered into 23 major clades containing multiple orthologous groups with single-copy gene from each species. We hypothesized that ORs in these orthologous groups are tuned to ecologically important compounds and functionally conserved. cis-Jasmone is one of the compounds that not only primes the plant defense of neighboring receiver plants, but also functions as a behavior regulator to various insects. To test our hypothesis, using Xenopus oocyte recordings, we functionally assayed the orthologues of BmorOR56, which has been characterized as a specific receptor for cis-jasmone. Our results showed highly conserved response specificity of the BmorOR56 orthologues, with all receptors within this group exclusively responding to cis-jasmone. This is supported by the dN/dS analysis, showing that strong purifying selection is acting on this group. Moreover, molecular docking showed that the ligand binding pockets of BmorOR56 orthologues to cis-jasmone are similar. Taken together, our results suggest the high conservation of OR for ecologically important compounds across Heterocera.

Cannabinoid CB2 receptor orthologues; in vitro function and perspectives for preclinical to clinical translation.

Cannabinoid CB2 receptor agonists are in development as therapeutic agents, including for immune modulation and pain relief. Despite promising results in rodent preclinical studies, efficacy in human clinical trials has been marginal to date. Fundamental differences in ligand engagement and signalling responses between the human CB2 receptor and preclinical model species orthologues may contribute to mismatches in functional outcomes. This is a tangible possibility for the CB2 receptor in that there is a relatively large degree of primary amino acid sequence divergence between human and rodent. Here, we summarise CB2 receptor gene and protein structure, assess comparative molecular pharmacology between CB2 receptor orthologues, and review the current status of preclinical to clinical translation for drugs targeted at the CB2 receptor, focusing on comparisons between human, mouse and rat receptors. We hope that raising wider awareness of, and proposing strategies to address, this additional challenge in drug development will assist in ongoing efforts toward successful therapeutic translation of drugs targeted at the CB2 receptor. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Evolutionary shifts in pheromone receptors contribute to speciation in four Helicoverpa species.

Male moths utilize their pheromone communication systems to distinguish potential mates from other sympatric species, which contributes to maintaining reproductive isolation and even drives speciation. The molecular mechanisms underlying the evolution of pheromone communication systems are usually studied between closely-related moth species for their similar but divergent traits associated with pheromone production, detection, and/or processing. In this study, we first identified the functional differentiation in two orthologous pheromone receptors, OR14b, and OR16, in four Helicoverpa species, Helicoverpa armigera, H. assulta, H. zea, and H. gelotopoeon. To understand the substrate response specificity of these two PRs, we performed all-atom molecular dynamics simulations of OR14b and OR16 based on AlphaFold2 structural prediction, andmolecular docking, allowing us to predict a few key amino acids involved in substrate binding. These candidate residues were further tested and validated by site-directed mutagenesis and functional analysis. These results together identified two hydrophobic amino acids at positions 164 and 232 are the determinants of the response specificity of HarmOR14b and HzeaOR14b to Z9-14:Ald and Z9-16:Ald by directly interacting with the substrates. Interestingly, in OR16 orthologs, we found that position 66 alone determines the specific binding of Z11-16:OH, likely via allosteric interactions. Overall, we have developed an effective integrated method to identify the critical residues for substrate selectivity of ORs and elucidated the molecular mechanism of the diversification of pheromone recognition systems.

Ancestral glycoprotein hormone-receptor pathway controls growth in C. elegans.

In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system in Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling show an increased defecation cycle period. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.

Development and Characterization of Potent Succinate Receptor Fluorescent Tracers.

The succinate receptor (SUCNR1) has emerged as a potential target for the treatment of various metabolic and inflammatory diseases, including hypertension, inflammatory bowel disease, and rheumatoid arthritis. While several ligands for this receptor have been reported, species differences in pharmacology between human and rodent orthologs have limited the validation of SUCNR1's therapeutic potential. Here, we describe the development of the first potent fluorescent tool compounds for SUCNR1 and use these to define key differences in ligand binding to human and mouse SUCNR1. Starting from known agonist scaffolds, we developed a potent agonist tracer, TUG-2384 (22), with affinity for both human and mouse SUCNR1. In addition, we developed a novel antagonist tracer, TUG-2465 (46), which displayed high affinity for human SUCNR1. Using 46 we demonstrate that three humanizing mutations on mouse SUCNR1, N181.31E, K2697.32N, and G84EL1W, are sufficient to restore high-affinity binding of SUCNR1 antagonists to the mouse receptor ortholog.

Prostanoid receptors in GtoPdb v.2023.1

Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [701]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Differences and similarities between human and rodent prostanoid receptor orthologues, and their specific roles in pathophysiologic conditions are reviewed in [452]. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.

Histamine receptors in GtoPdb v.2023.1

Histamine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Histamine Receptors [80, 174]) are activated by the endogenous ligand histamine. Marked species differences exist between histamine receptor orthologues [80]. The human and rat H3 receptor genes are subject to significant splice variance [12]. The potency order of histamine at histamine receptor subtypes is H3 = H4 > H2 > H1 [174]. Some agonists at the human H3 receptor display significant ligand bias [183]. Antagonists of all 4 histamine receptors have clinical uses: H1 antagonists for allergies (e.g. cetirizine), H2 antagonists for acid-reflux diseases (e.g. ranitidine), H3 antagonists for narcolepsy (e.g. pitolisant/WAKIX; Registered) and H4 antagonists for atopic dermatitis (e.g. adriforant; Phase IIa) [174] and vestibular neuritis (AUV) (SENS-111 (Seliforant, previously UR-63325), entered and completed vestibular neuritis (AUV) Phase IIa efficacy and safety trials, respectively) [217, 8].

Cannabidiol Recovers Dopaminergic Neuronal Damage Induced by Reserpine or α-synuclein in Caenorhabditis elegans.

Progressive neurodegenerative disorders such as Parkinson Disease (PD) lack curative or long-term treatments. At the same time, the increase of the worldwide elderly population and, consequently, the extension in the prevalence of age-related diseases have promoted research interest in neurodegenerative disorders. Caenorhabditis elegans is a free-living nematode widely used as an animal model in studies of human diseases. Here we evaluated cannabidiol (CBD) as a possible neuroprotective compound in PD using the C. elegans models exposed to reserpine. Our results demonstrated that CBD reversed the reserpine-induced locomotor alterations and this response was independent of the NPR-19 receptors, an orthologous receptor for central cannabinoid receptor type 1. Morphological alterations of cephalic sensilla (CEP) dopaminergic neurons indicated that CBD also protects neurons from reserpine-induced degeneration. That is, CBD attenuates the reserpine-induced increase of worms with shrunken soma and dendrites loss, increasing the number of worms with intact CEP neurons. Finally, we found that CBD also reduced ROS formation and α-syn protein accumulation in mutant worms. Our findings collectively provide new evidence that CBD acts as neuroprotector in dopaminergic neurons, reducing neurotoxicity and α-syn accumulation highlighting its potential in the treatment of PD.

Morphogenic Effect of Exogenous Glucocorticoid Hormones in the Girardia tigrina Planarian (Turbellaria, Tricladida).

We have studied the effect of two glucocorticoid hormones: hydrocortisone and its synthetic analogue methylprednisolone on the regeneration activity of head and tail blastema of the Girardia tigrina planarian. The regeneration activity was studied in head and tail blastema formed after resection by means of lifetime computer morphometry and immunohistochemical labeling of neoblasts. The search for orthologous proteins-glucocorticoid receptors (hydrocortisone) was performed using the SmedGD database of the Schmidtea mediterranea planarian. The results indicate that both hormones influence the recovery rate of the regenerating head and tail blastema. The worms with regenerating tail blastema have less sensitivity to the hormones' treatment compared to the ones with regenerating head blastema. Hydrocortisone at a high concentration (10-3 M) suppressed the regeneration rate, while stimulating it at lower concentrations (10-4-10-6 M). The same concentrations of methylprednisolone inhibited the regeneration of head blastema, but did not affect the tail blastema regeneration. The two hormones acted differently: while hydrocortisone stimulated the proliferation of neoblasts in the periwound region, methylprednisolone reduced the mitotic activity, mainly on the tail zone furthest from the wound surface. We suggest that exogenous glucocorticoids can influence endogenous mechanisms of hormone-dependent regeneration.

Antinociceptive Activity of Vanilloids in Caenorhabditis elegans is Mediated by the Desensitization of the TRPV Channel OCR-2 and Specific Signal Transduction Pathways.

Vanilloids, including capsaicin and eugenol, are ligands of transient receptor potential channel vanilloid subfamily member 1 (TRPV1). Prolonged treatment with vanilloids triggered the desensitization of TRPV1, leading to analgesic or antinociceptive effects. Caenorhabditis elegans (C. elegans) is a model organism expressing vanilloid receptor orthologs (e.g., OSM-9 and OCR-2) that are associated with behavioral and physiological processes, including sensory transduction. We have shown that capsaicin and eugenol hamper the nocifensive response to noxious heat in C. elegans. The objective of this study was to perform proteomics to identify proteins and pathways responsible for the induced phenotype and to identify capsaicin and eugenol targets using a thermal proteome profiling (TPP) strategy. The results indicated hierarchical differences following Reactome Pathway enrichment analyses between capsaicin- and eugenol-treated nematodes. However, both treated groups were associated mainly with signal transduction pathways, energy generation, biosynthesis and structural processes. Wnt signaling, a specific signal transduction pathway, is involved following treatment with both molecules. Wnt signaling pathway is noticeably associated with pain. The TPP results show that capsaicin and eugenol target OCR-2 but not OSM-9. Further protein-protein interaction (PPI) analyses showed other targets associated with enzymatic catalysis and calcium ion binding activity. The resulting data help to better understand the broad-spectrum pharmacological activity of vanilloids.

Comparative transcriptomic assessment of the chemosensory receptor repertoire of Drosophila suzukii adult and larval olfactory organs

The spotted wing Drosophila, Drosophila suzukii, has emerged within the past decade as an invasive species on a global scale, and is one of the most economically important pests in fruit and berry production in Europe and North America. Insect ecology, to a strong degree, depends on the chemosensory modalities of smell and taste. Extensive research on the sensory receptors of the olfactory and gustatory systems in Drosophila melanogaster provide an excellent frame of reference to better understand the fundamentals of the chemosensory systems of D. suzukii. This knowledge may enhance the development of semiochemicals for sustainable management of D. suzukii, which is urgently needed. Here, using a transcriptomic approach we report the chemosensory receptor expression profiles in D. suzukii female and male antennae, and for the first time, in larval heads including the dorsal organ that houses larval olfactory sensory neurons. In D. suzukii adults, we generally observed a lack of sexually dimorphic expression levels in male and female antennae. While there was generally conservation of antennal expression of odorant and ionotropic receptor orthologues for D. melanogaster and D. suzukii, gustatory receptors showed more distinct species-specific profiles. In larval head tissues, for all three receptor gene families, there was also a greater degree of species-specific gene expression patterns. Analysis of chemosensory receptor repertoires in the pest species, D. suzukii relative to those of the genetic model D. melanogaster enables comparative studies of the chemosensory, physiology, and ecology of D. suzukii.

Bitter taste sensitivity in domestic dogs (Canis familiaris) and its relevance to bitter deterrents of ingestion.

As the most favoured animal companion of humans, dogs occupy a unique place in society. Understanding the senses of the dog can bring benefits to both the dogs themselves and their owners. In the case of bitter taste, research may provide useful information on sensitivity to, and acceptance of, diets containing bitter tasting materials. It may also help to protect dogs from the accidental ingestion of toxic substances, as in some instances bitter tasting additives are used as deterrents to ingestion. In this study we examined the receptive range of dog bitter taste receptors (Tas2rs). We found that orthologous dog and human receptors do not always share the same receptive ranges using in vitro assays. One bitter chemical often used as a deterrent, denatonium benzoate, is only moderately active against dTas2r4, and is almost completely inactive against other dog Tas2rs, including dTas2r10, a highly sensitive receptor in humans. We substituted amino acids to create chimeric dog-human versions of the Tas2r10 receptor and found the ECL2 region partly determined denatonium sensitivity. We further confirmed the reduced sensitivity of dogs to this compound in vivo. A concentration of 100μM (44.7ppm) denatonium benzoate was effective as a deterrent to dog ingestion in a two-bottle choice test indicating higher concentrations may increase efficacy for dogs. These data can inform the choice and concentration of bitter deterrents added to toxic substances to help reduce the occurrence of accidental dog poisonings.

Molecular and Pharmacological Characterization of β-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae)

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three β-adrenergic-like OARs (CcOctβRs) from Cotesia chilonis. CcOctβRs share high similarity with their own orthologous receptors. The transcript levels of CcOctβRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctβRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of β-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

Molecular characterization, targeting and expression analysis of chloroplast and mitochondrion protein import components in Nicotiana benthamiana.

Improved bioinformatics tools for annotating gene function are becoming increasingly available, but such information must be considered theoretical until further experimental evidence proves it. In the work reported here, the genes for the main components of the translocons of the outer membrane of chloroplasts (Toc) and mitochondria (Tom), including preprotein receptors and protein-conducting channels of N. benthamiana, were identified. Sequence identity searches and phylogenetic relationships with functionally annotated sequences such as those of A. thaliana revealed that N. benthamiana orthologs mainly exist as recently duplicated loci. Only a Toc34 ortholog was found (NbToc34), while Toc159 receptor family was composed of four orthologs but somewhat different from those of A. thaliana. Except for NbToc90, the rest (NbToc120, NbToc159A and NbToc159B) had a molecular weight of about 150 kDa and an acidic domain similar in length. Only two orthologs of the Tom20 receptors, NbTom20-1 and NbTom20-2, were found. The number of the Toc and Tom receptor isoforms in N. benthamiana was comparable to that previously reported in tomato and what we found in BLAST searches in other species in the genera Nicotiana and Solanum. After cloning, the subcellular localization of N. benthamiana orthologs was studied, resulting to be identical to that of A. thaliana receptors. Phenotype analysis after silencing together with relative expression analysis in roots, stems and leaves revealed that, except for the Toc and Tom channel-forming components (NbToc75 and NbTom40) and NbToc34, functional redundancy could be observed either among Toc159 or mitochondrial receptors. Finally, heterodimer formation between NbToc34 and the NbToc159 family receptors was confirmed by two alternative techniques indicating that different Toc complexes could be assembled. Additional work needs to be addressed to know if this results in a functional specialization of each Toc complex.