The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50 nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2–T1R3. Chimeric human– mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2– mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.     

Metabotropic glutamate receptors and neuroadaptation to antidepressants: imipramine-induced down-regulation of beta-adrenergic receptors in mice treated with metabotropic glutamate 2/3 receptor ligands

Antidepressant drugs have a clinical latency that correlates with the development of neuroadaptive changes, including down-regulation of beta-adrenergic receptors in different brain regions. The identification of drugs that shorten this latency will have a great impact on the treatment of major depressive disorders. We report that the time required for the antidepressant imipramine to reduce the expression of beta-adrenergic receptors in the hippocampus is reduced by a co-administration with centrally active ligands of type 2/3 metabotropic glutamate (mGlu2/3) receptors. Daily treatment of mice with imipramine alone (10 mg/kg, i.p.) reduced the expression of beta-adrenergic receptors in the hippocampus after 21 days, but not at shorter times, as assessed by western blot analysis of beta1-adrenergic receptors and by the amount of specifically bound [3H]CGP-12177, a selective beta-adrenergic receptor ligand. Down-regulation of beta-adrenergic receptors occurred at shorter times (i.e. after 14 days) when imipramine was combined with low doses (0.5 mg/kg, i.p.) of the selective mGlu2/3 receptor agonist LY379268, or with the preferential mGlu2/3 receptor antagonist LY341495 (1 mg/kg, i.p.). Higher doses of LY379268 (2 mg/kg, i.p.) were inactive. This intriguing finding suggests that neuroadaptation to imipramine--at least as assessed by changes in the expression of beta1-adrenergic receptors--is influenced by drugs that interact with mGlu2/3 receptors and stimulates further research aimed at establishing whether any of these drugs can shorten the clinical latency of classical antidepressants.     

Deficiency of P-selectin or P-selectin glycoprotein ligand-1 leads to accelerated development of glomerulonephritis and increased expression of CC chemokine ligand 2 in lupus-prone mice

The selectins and their ligands mediate leukocyte rolling on endothelial cells, the initial step in the emigration cascade leading to leukocyte infiltration of tissue. These adhesion molecules have been shown to be key promoters of acute leukocyte emigration events; however, their roles in the development of long-term inflammatory responses, including those that occur during chronic inflammatory diseases such as systemic lupus erythematosus, are unclear. To assess participation of P-selectin in such disorders, we studied the progression of systemic lupus erythematosus-like disease in P-selectin-deficient and control MRL/MpJ-Fas(lpr) (Fas(lpr)) mice. Surprisingly, we found that P-selectin deficiency resulted in significantly earlier mortality, characterized by a more rapid development of glomerulonephritis and dermatitis. Expression of CCL2 (MCP-1) was increased in the kidneys of P-selectin mutant mice and in supernatants of LPS-stimulated primary renal endothelial cell cultures from these mice. A closely similar phenotype, including elevated renal expression of CCL2, was also observed in Fas(lpr) mice deficient in the major P-selectin ligand, P-selectin glycoprotein ligand-1. These results indicate that P-selectin and P-selectin glycoprotein ligand-1 are not required for leukocyte infiltration and the development of autoimmune disease in Fas(lpr) mice, but rather expression of these adhesion molecules is important for modulating the progression of glomerulonephritis, possibly through down-regulation of endothelial CCL2 expression.     

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Anti-inflammatory effects of inosine in allergic lung inflammation in mice: evidence for the participation of adenosine A2A and A3 receptors

Inosine, a naturally occurring purine formed from the breakdown of adenosine, is associated with immunoregulatory effects. Evidence shows that inosine modulates lung inflammation and regulates cytokine generation. However, its role in controlling allergen-induced lung inflammation has yet to be identified. In this study, we aimed to investigate the role of inosine and adenosine receptors in a murine model of lung allergy induced by ovalbumin (OVA). Intraperitoneal administration of inosine (0.001–10 mg/kg, 30 min before OVA challenge) significantly reduced the number of leukocytes, macrophages, lymphocytes and eosinophils recovered in the bronchoalveolar lavage fluid of sensitized mice compared with controls. Interestingly, our results showed that pre-treatment with the selective A_2A receptor antagonist (ZM241385), but not with the selective A_2B receptor antagonist (alloxazine), reduced the inhibitory effects of inosine against macrophage count, suggesting that A_2A receptors mediate monocyte recruitment into the lungs. In addition, the pre-treatment of mice with selective A₃ antagonist (MRS3777) also prevented inosine effects against macrophages, lymphocytes and eosinophils. Histological analysis confirmed the effects of inosine and A_2A adenosine receptors on cell recruitment and demonstrated that the treatment with ZM241385 and alloxazine reverted inosine effects against mast cell migration into the lungs. Accordingly, the treatment with inosine reduced lung elastance, an effect related to A₂ receptors. Moreover, inosine reduced the levels of Th₂-cytokines, interleukin-4 and interleukin-5, an effect that was not reversed by A_2A or A_2B selective antagonists. Our data show that inosine acting on A_2A or A₃ adenosine receptors can regulate OVA-induced allergic lung inflammation and also implicate inosine as an endogenous modulator of inflammatory processes observed in the lungs of asthmatic patients.     

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.     

A single dose of methamphetamine leads to a long term reversal of the blunted dopamine D1 receptor-mediated neocortical c-fos responses in mice deficient for D2 and D3 receptors

Dopamine D(1) receptors play an essential role in the induction of expression of the immediate-early gene c-fos in response to pharmacological stimuli. In the forebrain of wild-type mice, administration of a D(1) receptor agonist leads to c-fos mRNA expression levels that are substantially higher than corresponding levels expressed after indirect stimulation of dopamine receptors with methamphetamine. In mice deficient for D(2) and D(3) receptors, c-fos mRNA levels expressed in response to D(1) agonist administration are significantly blunted. However, a single dose of methamphetamine (5 mg/kg) leads to a long lasting reversal of the blunted c-fos responses in these mutants. In the forebrain, this reversal is restricted to the neocortex. Moreover, methamphetamine also enhances c-fos expression levels in preadolescent wild-type mice that normally express low c-fos mRNA in response to D(1) agonist stimulation. Thus, a single dose of methamphetamine leads to a long term increase in D(1) receptor-dependent c-fos responses in brains with either low (preadolescent mice) or blunted (adult D(2) and D(3) mutant mice) c-fos expression levels. A similar long term reversal of the blunted c-fos responses is achieved with a single dose of a full D(1) agonist. These results indicate that the constitutive inactivation of D(2) and D(3) receptors leads to a decrease in agonist-promoted D(1) receptor activity that can be reversed by intermittent agonist stimulation.     

Age- and sex-dependent effect of exogenous melatonin on expression pattern of melatonin receptor (MT1 and MT2) proteins in spleen of mice

Spleen is an important lymphoid organ which exerts immune activities throughout the life in mammals. In this study, we investigated the age- and sex-dependent effect of exogenous melatonin on expression pattern of MT1 and MT2 melatonin receptor proteins in spleen of laboratory Swiss albino mice in three different age-groups – 2, 4, and 8 months. The melatonin receptor expression patterns were studied by immunohistochemical localization and Western blot analysis. Immunohistochemical study showed reactivity of MT1 and MT2 melatonin receptors in spleen of both male and female mice. Exogenous melatonin significantly showed age- and sex-dependent expression pattern of MT1 receptor protein, while MT2 receptors showed only age-dependent differential expression patterns in both male and female mice. Therefore, this study may suggest that exogenous melatonin is modulating MT1 and MT2 receptor protein expression pattern in age- and sex-dependent manner in spleen of mice.     

Role of the MT1 and MT2 melatonin receptors in mediating depressive‐ and anxiety‐like behaviors in C3H/HeN mice

Melatonin is a neurohormone primarily synthesized by the pineal gland following a circadian rhythm with a high level during the night and a low level during the day. Alterations in the synthesis and secretion of melatonin have been reported in various mood disorders, including major depressive disorder. However, the role of endogenous melatonin in the pathophysiology of depressive disorder is unclear. Melatonin primarily acts through two G protein‐coupled receptors, termed MT₁ and MT₂. The present study investigated the effect of genetic deletion of the MT₁ and/or MT₂ receptors on tests associated with depression‐ and anxiety‐like behaviors in C3H/HeN mice. Deletion of the MT₁ and/or MT₂ receptors caused a deficit in hedonic and social interaction behavior, and increased anxiety‐like behavior. It is likely that dysregulations of the MT₁ and/or MT₂ melatonin receptors could be involved in the pathophysiology of depression and anxiety.     

Adenosine A2A receptors are required for glutamate mGluR5‐ and dopamine D1 receptor‐evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor

Interaction between mGluR5 and NMDA receptors (NMDAR ) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A_2A receptors (A_2AR) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDAR s co‐stimulation synergistically activate ERK 1/2 signaling leading to c‐Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A_2ARs. Moreover, mGluR5‐mediated ERK 1/2 phosphorylation depends on NMDAR , which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR ‐evoked ERK 1/2 activation correlates well with the mGluR5/NMDAR ‐evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A_2ARs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK 1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA 1 region of hippocampus, the theta burst stimulation (TBS)‐induced long‐term potentiation coincides temporally with an increase in ERK 1/2 activation and both phenomena are dependent on the tripartite A_2A, mGlu5, and NMDAR s. Furthermore, we show that the dopamine D1 receptors evoked ERK 1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A_2ARs. In conclusion, our results highlight the A_2ARs as a crucial regulator not only for NMDAR responses, but also for regulating ERK 1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation.

     

APLP1 and APLP2, members of the APP family of proteins,behave similarly to APP in that they associate with NMDA receptors and enhance NMDA receptor surface expression

The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d ‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.

     

Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC‐84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force‐generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1‐1 Atsun2‐2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock‐like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.     

Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark,Squalus acanthias

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D₃ versus D₂ receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (K_i) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D₂ or D₃ receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D₃ affinity whereas the indole derivative 13 exhibited similar high D₃ affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D₃ affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization.     

Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB(1)), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB(1) antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB(1) gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB(1) distribution only in part. In CB(1) knockout homozygotes (CB(1)-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB(1)+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB(1) in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB(1)-/- with respect to CB(1)+/+ mice in all regions. AEA and Delta(9)-tetrahydrocannabinol (THC) were tested in CB(1)-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB(1)-/- mice. AEA, but not THC, stimulated GTPgammaS binding in brain membranes from CB(1)-/- mice, and this stimulation was insensitive to CB(1) and CB(2) antagonists. We suggest that non-CB(1), non-CB(2) G protein-coupled receptors might mediate in mice some of the neuro-behavioral actions of AEA.     

Binding characteristics and sensitivity to endogenous dopamine of [11C]-(+)-PHNO, a new agonist radiotracer for imaging the high-affinity state of D2 receptors in vivo using positron emission tomography

[11C]-(+)-PHNO (4-propyl-9-hydroxynaphthoxazine) is a new agonist radioligand that provides a unique opportunity to measure the high-affinity states of the D2 receptors (D2-high) using positron emission tomography (PET). Here we report on the distribution, displaceablity, specificity and modeling of [11C]-(+)-PHNO and compare it with the well characterized antagonist D2 radioligand, [11C]raclopride, in cat. [11C]-(+)-PHNO displayed high uptake in striatum with a mean striatal binding potential (BP) of 3.95 +/- 0.85. Pre-treatment with specific D1 (SCH23390), D2 (raclopride, haloperidol) and D3 receptor (SB-277011) antagonists indicated that [11C]-(+)-PHNO binding in striatum is specific to D2 receptors. Within-subject comparisons showed that [11C]-(+)-PHNO BP in striatum was almost 2.5-fold higher than that measured with [11C]-(-)-NPA ([11C]-(-)-N-propyl-norapomorphine). Comparison of the dose-effect of amphetamine (0.1, 0.5 and 2 mg/kg; i.v.) showed that [11C]-(+)-PHNO was more sensitive to the dopamine releasing effect of amphetamine than [11C]raclopride. Amphetamine induced up to 83 +/- 4% inhibition of [11C]-(+)-PHNO BP and only up to 56 +/- 8% inhibition of [11C]raclopride BP. Scatchard analyses of [11C]-(+)-PHNO and [11C]raclopride bindings in two cats showed that the Bmax obtained with the agonist (29.6 and 32.9 pmol/mL) equalled that obtained with the antagonist (30.6 and 33.4 pmol/mL). The high penetration of [11C]-(+)-PHNO in brain, its high signal-to-noise ratio, its favorable in vivo kinetics and its high sensitivity to amphetamine shows that [11C]-(+)-PHNO has highly suitable characteristics for probing the D2-high with PET.     

But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast

NEDD8/Rub1 is the most homologous protein to ubiquitin among the ubiquitin-like proteins, and it is covalently linked to target proteins via the C-terminal glycine residue in a manner analogous to ubiquitylation. However, the mechanism(s) involved in the regulation of the NEDD8 ligation pathway remains elusive. Using the two-hybrid system, we isolated novel genes from the Schizosaccharomyces pombe cDNA library whose products bind to Uba3, which is a catalytic protein for E1-like activity of the NEDD8 pathway. We designated these genes but1(+) and but2(+) (for proteins that bind to Uba three). But1 is a nuclear protein and its overexpression caused cell elongation, which is a common phenotype of the NEDD8 pathway defective mutant in S. pombe. Furthermore, overexpression of but1(+) in ned8-temperature sensitive mutant had a deleterious effect even under permissive temperatures. Our results suggest that But1 may have an inhibitory role in the NEDD8 pathway.