Generation and identification of kokumi compounds and their validation by taste-receptor assay: An example with dry-cured lamb meat.

Kokumi tastants are small γ-glutamyl peptides (GGP) that enhance flavour in foods. We sought to generate GGP from the meat crusts of dry-cured lamb, an underutilised protein resource, identify these using mass spectrometry, and validate their functional activity using a kokumi-calcium sensing receptor (CaSR) assay. The water-soluble extract (WSE) of meat crust was hydrolysed by protease A (PA) and treated with glutaminase (GA). Fifteen GGP were identified, with 14 being significantly increased in PA and GA groups compared to WSE, as were along with free amino acid levels. The GA extract activated CaSR with higher potency and efficacy than PA and WSE suggesting the generation of potent kokumi tastants. The in vitro receptor assay might be an expedient tool for screening kokumi tastants prior to conducting human sensory analysis. Collectively, our findings indicate that the meat crust can be a valuable source to generate kokumi tastants via a two-step enzymatic reaction.

Objective Measurements Associated with the Preferred Eating Qualities of Fermented Salamis.

The development of new food products can be expedited by understanding the physicochemical attributes that are most relevant to consumers. Although many objective analyses are possible, not all are a suitable proxy to serve as quality markers associated with sensory preferences. In this work, we selected nine candidate laboratory assays to use on six commercial salamis, which were also eaten and informally described by a consumer discussion group familiar with China-sourced meat products. Several objective measures were strongly related to the flavour perceptions: (i) texture: instrumental texture values, fat release at oral temperature and fat saturation ratios, (ii) aroma: volatile compounds (e.g., alcohols and esters) associated with microbial fermentation and spices (terpenes and sulphur compounds) and (iii) taste: kokumi taste receptor responses. The fat released at oral temperature was associated with unsaturated fatty acids (r = 0.73). However, there was less explanatory worth for associations between sensory perceptions and proximate composition, water activity, pH or L*, a*, b* colourimetry.

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins.

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and ß-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT1 and MT2 receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of ß-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT1 is only mediated though Gi/o proteins, while MT2 is dependent on the cooperative activation of Gi/o and Gq/11 proteins. In the absence of Gq/11 proteins, however, MT2 -induced ERK1/2 activation switches to a ß-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT1 - and MT2 -mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. Gi/o /Gq/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT2 , whereas in the mouse retina, where MT2 is engaged into MT1 /MT2 heterodimers, ERK1/2 signaling is exclusively Gi/o -dependent. Collectively, our data reveal differential signaling modes of MT1 and MT2 in terms of ERK1/2 activation, with an unexpected Gi/o /Gq/11 cooperativity exclusively for MT2 . The plasticity of ERK activation by MT2 is highlighted by the switch to a ß-arrestin1/2-dependent mode in the absence of Gq/11 proteins and by the switch to a Gi/o mode when engaged into MT1 /MT2 heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

G Protein-Coupled Receptors in Taste Physiology and Pharmacology.

Heterotrimeric G protein-coupled receptors (GPCRs) comprise the largest receptor family in mammals and are responsible for the regulation of most physiological functions. Besides mediating the sensory modalities of olfaction and vision, GPCRs also transduce signals for three basic taste qualities of sweet, umami (savory taste), and bitter, as well as the flavor sensation kokumi. Taste GPCRs reside in specialised taste receptor cells (TRCs) within taste buds. Type I taste GPCRs (TAS1R) form heterodimeric complexes that function as sweet (TAS1R2/TAS1R3) or umami (TAS1R1/TAS1R3) taste receptors, whereas Type II are monomeric bitter taste receptors or kokumi/calcium-sensing receptors. Sweet, umami and kokumi receptors share structural similarities in containing multiple agonist binding sites with pronounced selectivity while most bitter receptors contain a single binding site that is broadly tuned to a diverse array of bitter ligands in a non-selective manner. Tastant binding to the receptor activates downstream secondary messenger pathways leading to depolarization and increased intracellular calcium in TRCs, that in turn innervate the gustatory cortex in the brain. Despite recent advances in our understanding of the relationship between agonist binding and the conformational changes required for receptor activation, several major challenges and questions remain in taste GPCR biology that are discussed in the present review. In recent years, intensive integrative approaches combining heterologous expression, mutagenesis and homology modeling have together provided insight regarding agonist binding site locations and molecular mechanisms of orthosteric and allosteric modulation. In addition, studies based on transgenic mice, utilizing either global or conditional knock out strategies have provided insights to taste receptor signal transduction mechanisms and their roles in physiology. However, the need for more functional studies in a physiological context is apparent and would be enhanced by a crystallized structure of taste receptors for a more complete picture of their pharmacological mechanisms.

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain.

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24-hour sleep-wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR-KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.

Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.

The orphan GPR50 receptor promotes constitutive TGFß receptor signaling and protects against cancer development.

Transforming growth factor-ß (TGFß) signaling is initiated by the type I, II TGFß receptor (TßRI/TßRII) complex. Here we report the formation of an alternative complex between TßRI and the orphan GPR50, belonging to the G protein-coupled receptor super-family. The interaction of GPR50 with TßRI induces spontaneous TßRI-dependent Smad and non-Smad signaling by stabilizing the active TßRI conformation and competing for the binding of the negative regulator FKBP12 to TßRI. GPR50 overexpression in MDA-MB-231 cells mimics the anti-proliferative effect of TßRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV/Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer irrespective of the breast cancer subtype. This describes a previously unappreciated spontaneous TGFß-independent activation mode of TßRI and identifies GPR50 as a TßRI co-receptor with potential impact on cancer development.

Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT2 receptor reciprocally modulate their signaling functions.

Understanding the function of orphan G protein-coupled receptors (GPCRs), whose cognate ligand is unknown, is of major importance as GPCRs are privileged drug targets for many diseases. Recent phylogenetic studies classified three orphan receptors, GPR61, GPR62 and GPR135 among the melatonin receptor subfamily, but their capacity to bind melatonin and their biochemical functions are not well characterized yet. We show here that GPR61, GPR62 and GPR135 do not bind [3H]-melatonin nor 2-[125I]iodomelatonin and do not respond to melatonin in several signaling assays. In contrast, the three receptors show extensive spontaneous ligand-independent activities on the cAMP, inositol phosphate and ß-arrestin pathways with distinct pathway-specific profiles. Spontaneous ß-arrestin recruitment internalizes all three GPRs in the endosomal compartment. Co-expression of the melatonin binding MT2 receptor with GPR61, GPR62 or GPR135 has several consequences such as (i) the formation of receptor heteromers, (ii) the inhibition of melatonin-induced ß-arrestin2 recruitment to MT2 and (iii) the decrease of elevated cAMP levels upon melatonin stimulation in cells expressing spontaneously active GPR61 and GPR62. Collectively, these data show that GPR61, GPR62 and GPR135 are unable to bind melatonin, but show a reciprocal regulatory interaction with MT2 receptors.

Hunting for the function of orphan GPCRs - beyond the search for the endogenous ligand.

Seven transmembrane-spanning proteins (7TM), also called GPCRs, are among the most versatile and evolutionary successful protein families. Out of the 400 non-odourant members identified in the human genome, approximately 100 remain orphans that have not been matched with an endogenous ligand. Apart from the classical deorphanization strategies, several alternative strategies provided recent new insights into the function of these proteins, which hold promise for high therapeutic potential. These alternative strategies consist of the phenotypical characterization of organisms silenced or overexpressing orphan 7TM proteins, the search for constitutive receptor activity and formation of protein complexes including 7TM proteins as well as the development of synthetic, surrogate ligands. Taken together, a variety of ligand-independent functions can be attributed to orphan 7TM proteins that range from constitutive activity to complex formation with other proteins and include 'true' orphans for which no ligand exist and 'conditional' orphans that behave like orphans in the absence of ligand and as non-orphans in the presence of ligand.

Photoperiodic regulation of nuclear melatonin receptor RORα in lymphoid organs of a tropical rodent Funambulus pennanti: role in seasonal oxidative stress.

The orphan nuclear receptor RORα of RZR/ROR family has been suggested to mediate the genomic actions of melatonin on the expression of antioxidant enzymes. However, no direct evidences exist. In the present study we explored the role of photoperiod (natural and artificial) in regulation of RORα and its association with the photoperiod induced antioxidant defense system in the lymphoid organs (spleen and thymus) of seasonally breeding, tropical squirrels, Funambulus pennanti. The photoperiod mediated regulation of antioxidant status was checked along with the RORα expression and circulatory melatonin level in the squirrels. The enhancement of the antioxidant capacity of serum and lymphoid organ was concomitant with the short photoperiod (10L:14D) induced high levels of plasma melatonin. Further, peripheral melatonin level enhanced the AANAT activity as well as the melatonin synthesis in the lymphoid tissues. RORα expression presented an inverse correlation with the plasma level of melatonin as well as the short day induced antioxidant enzyme activity in the lymphoid organs. The results suggest that for reduction of seasonal oxidative stress melatonin might not be utilizing the nuclear receptor RORα pathway; rather the rise in circulatory melatonin collectively with tissue specific melatonin might be protecting the splenic and thymic lymphocytes from the seasonal oxidative stress.

Immune responses to lipopolysaccharide challenge in a tropical rodent (Funambulus pennanti): photoperiod entrainment and sex differences.

Annual variation in day length (photoperiod) triggers changes in the immune system of seasonal breeders. The rationale behind this study was to delineate any sex differences in immune responses of photoperiodically entrained animals challenged against lipopolysaccharide (LPS)-induced inflammatory stress. We observed that photoperiodically entrained [short day, SD, 10 h light (L):14 h dark (D); long day, LD, 16 h L:8 h D; and natural day length, NDL, 12 h L:12 h D] male and female Indian palm squirrels, Funambulus pennanti, presented sexual dimorphism in immune status after LPS-induced stress. Females presented high humoral (anti-keyhole limpet hemocyanin immunoglobulin) and cellular immunity (lymphocyte proliferation) compared with the males of all photoperiodic conditions. Female squirrels showed reduced pro-inflammatory cytokine levels (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) than the males suggesting their high efficiency to recover from LPS-induced inflammatory stress. Increased duration of melatonin secretion and corticosterone concentration in squirrels experiencing SD evidently supported survival of squirrels as compared with control (NDL) and LD squirrels of both sexes. Decreased immune status in both sexes under LD condition might be due to a short melatonin signal mimicking the LDs of summer. Thus, we infer that photoperiodic entrainment via the levels of melatonin and corticosterone synergistically supported more the survival of female squirrels under LPS-induced stress.

Melatonin membrane receptor type MT1 modulates cell-mediated immunity in the seasonally breeding tropical rodent Funambulus pennanti.

OBJECTIVE: Despite the evidence for melatonin membrane receptors (MT1R and MT2R) on lymphoid tissues in a wide range of seasonal breeders, their specific potency has never been compared and correlated with cell-mediated immunity. METHODS: We used luzindole, a nonselective MT2R antagonist, and 4-phenyl-2-propionamidotetralin (4P-PDOT), a selective MT2R antagonist, to assess the potency of the melatonin receptors MT1 and MT2 in melatonin-induced immunity under both in vivo as well as in vitro conditions. RESULTS: Physiological doses (25 µg/100 g body weight in vivo and 100 and 500 pg/ml in vitro) of melatonin upregulated both MT1R and MT2R expression as well as splenocyte proliferation, while higher doses (100 and 500 µg/100 g body weight in vivo and 1 ng/ml in vitro) downregulated splenocyte proliferation and the expression of both receptors. Luzindole antagonized the expression of both MT1R and MT2R in a dose-dependent manner under in vivo as well as in vitro conditions, while 4P-PDOT blocked the expression of MT2R only during both experimental conditions. Splenocyte proliferation and IL-2 secretion (in vitro) followed the MT1R expression pattern, while the MT2R expression pattern showed no definite relation with either splenocyte proliferation or IL-2 secretion under in vivo and in vitro conditions. CONCLUSION: Immune function in tropical rodents is directly regulated by melatonin via its high-affinity membrane receptor MT1. MT1R plays a directive role in mediating splenocyte proliferation and IL-2 release, while the MT2R subtype appears not to be required for the immunoenhancing role of melatonin.

Age dependent expression of melatonin membrane receptor (MT1, MT2) and its role in regulation of nitrosative stress in tropical rodent Funambulus pennanti.

Age-dependent declining level of melatonin induces free radical load and thereby deteriorates immune function. However, reports are lacking about age-dependent melatonin membrane receptor (MT1 & MT2) expression, their role in regulation of reactive nitrogen species (RNS) and eventually how they affect immunity of a tropical rodent F. pennanti. We checked MT1R, MT2R and iNOS expression in lymphoid organs of young middle and old aged squirrels. Nitrite and nitrate ion concentration (NOx) in lymphoid organs, testes and plasma, lymphocyte proliferation and IL-2 level was recorded. Age-dependent decrease in MT1 and MT2 receptor expression, lymphocyte proliferation, IL-2 level and increased RNS in lymphoid organs, testes and plasma was observed with decreased circulatory melatonin. Androgen and AR expression was increased in middle-aged while declined in old-aged squirrels. Present study suggests that age associated immunosenescence is consequence of increased RNS which might have important relationship with melatonin membrane receptors in F. pennanti.

Effect of intra-testicular melatonin injection on testicular functions, local and general immunity of a tropical rodent Funambulus pennanti.

Local antigonadotrophic action of melatonin in testes has never been correlated with local and general immune status of any rodent. Intra-testicular injection of melatonin (2.5 µg/50 µl) for 10 days (MI-10D) and 20 days (MI-20D) was given to young adult male of Funambulus pennanti and testicular androgen receptor (AR), androgen binding protein (ABP) expression, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity, and Mel1aR expression in thymus was checked along with general immune parameters. Further, immunohistochemical localization of Mel1aR in testes was done. Decreased AR, ABP expression, testes weight, 3ß-HSD activity, testosterone level, and spermatogenesis but increased Mel1aR expression in thymus, immunoreactivity in testes, and testicular macrophages following injection was noted. Lymphatic tissue weight, leukocyte, lymphocyte count, lymphocyte proliferation in spleen, thymus, plasma melatonin, and IL-2 level increased in a duration-dependent manner following intra-testicular injection. Intra-testicular injection of melatonin decreased steroidogenesis by enhancing the primary effect of melatonin on Leydig cell endocrine function. Along with reduced circulatory testosterone production, an increase in testicular as well as general immunity was observed in a duration-dependent manner. Therefore, a local participation of melatonin in testes of F. pennanti to control testicular androgen production is suggested.

Photoperiodic regulation of MT1 and MT2 melatonin receptor expression in spleen and thymus of a tropical rodent Funambulus pennanti during reproductively active and inactive phases.

Photoperiodic regulation of melatonin receptor types on target tissues, such as lymphatic organs, has never been explored for any seasonal breeder. In the present study, we accessed the high affinity membrane melatonin receptors MT1 and MT2 expression dynamics in lymphoid organs (i.e., spleen and thymus) of a seasonally breeding rodent Funambulus pennanti during two major reproductive phases (i.e., active and inactive), when the internal hormonal (melatonin and gonadal steroid) as well as the ecological conditions were entirely different. Photoperiod regulates circulatory melatonin level; hence, we noted the effect of different photoperiodic regimes (long; 16L:8D and short; 10L:14D photoperiod) equivalent to summer and winter daylength on membrane melatonin receptor MT1 and MT2 expression in spleen and thymus. We have correlated the melatonin receptor expression with two major hormones varying seasonally (i.e., melatonin and testosterone) also being responsible for modulation of immunity of a seasonal breeder. Differential immunoreactivity of MT1 and MT2 receptor in spleen and thymus of F. pennanti suggests an involvement of both the receptor types in signal transduction of photoperiod for seasonal immunomodulation, because in the tropical zone, a slight difference (1:45-2 h) in daylength may change reproductive physiology and immunity of animals for adaptation. Our above suggestion receives strong support from the experiment of photoperiodic exposure on MT1 and MT2 expression at the translational level, where long daylength decreased the circulatory melatonin level and melatonin receptor expression in both lymphatic tissues. On the other hand, under short daylength, expression of MT1 and MT2 receptor increased in both spleen and thymus along with concomitant increase in circulatory melatonin level. Differential hormonal level of melatonin and gonadal hormones during reproductively active and inactive phase and its direct relation with melatonin receptor expression dynamics in lymphoid organs could be responsible for seasonal adjustment of immunity and reproduction.