Unveiling the transcriptomic landscape and the potential antagonist feedback mechanisms of TGF-ß superfamily signaling module in bone and osteoporosis.

BACKGROUND: TGF-ß superfamily signaling is indispensable for bone homeostasis. However, the global expression profiles of all the genes that make up this signaling module in bone and bone-related diseases have not yet been well characterized. METHODS: Transcriptomic datasets from human bone marrows, bone marrow-derived mesenchymal stem cells (MSCs) and MSCs of primary osteoporotic patients were used for expression profile analyses. Protein treatments, gene quantification, reporter assay and signaling dissection in MSC lines were used to clarify the interactive regulations and feedback mechanisms between TGF-ß superfamily ligands and antagonists. Ingenuity Pathway Analysis was used for network construction. RESULTS: We identified TGFB1 in the ligand group that carries out SMAD2/3 signaling and BMP8A, BMP8B and BMP2 in the ligand group that conducts SMAD1/5/8 signaling have relatively high expression levels in normal bone marrows and MSCs. Among 16 antagonist genes, the dominantly expressed TGF-ß superfamily ligands induced only NOG, GREM1 and GREM2 via different SMAD pathways in MSCs. These induced antagonist proteins further showed distinct antagonisms to the treated ligands and thus would make up complicated negative feedback networks in bone. We further identified TGF-ß superfamily signaling is enriched in MSCs of primary osteoporosis. Enhanced expression of the genes mediating TGF-ß-mediated SMAD3 signaling and the genes encoding TGF-ß superfamily antagonists served as significant features to osteoporosis. CONCLUSION: Our data for the first time unveiled the transcription landscape of all the genes that make up TGF-ß superfamily signaling module in bone. The feedback mechanisms and regulatory network prediction of antagonists provided novel hints to treat osteoporosis. Video Abstract.

Expression profiling of ovarian BMP antagonists reveals the potential interaction between TWSG1 and the chordin subfamily in the ovary.

The TGF-ß superfamily members and their antagonists comprise an indispensable system that controls mammalian ovarian development in a sophisticated manner. In contrast to a plethora of studies on the ovary-expressed TGF-ß superfamily members, knowledge regarding their antagonists, including their expression profiles and antagonism preferences, is still lacking. Using quantitative PCR in rats and transcriptomic dataset comparisons in mice and humans, we set out to characterize the relative expression levels of most antagonists in the mammalian ovary. We found that Twsg1 and Nbl1 are the most abundant BMP antagonists expressed in the rodent and human ovaries, respectively. TWSG1 has been reported to have synergistic action with the chordin subfamily, including CHRD and CHRDL1, the genes of which also showed moderate expression in the mammalian ovary. Therefore, their ovarian expression profiles and antagonisms against the ovary-expressed TGF-ß superfamily members were further characterized. Bioactivity tests indicated that TWSG1 alone can directly inhibit the signaling of BMP6 or BMP7. In addition, it can further enhance the antagonizing ability of CHRD towards BMP2, BMP4, BMP7 and GDF5, or CHRDL1's antagonism towards BMP2, BMP4, GDF5 and activin A. In combination with their distinct transcript profiles in ovarian compartments, our findings suggest that TWSG1 may work coordinately with CHRD within theca/interstitial shells and also with CHRDL1 in developing granulosa cells; these interactions would modulate the intraovarian functions of the TGF-ß superfamily members, such as the control of progesterone production.

Human bone morphogenetic protein 8A promotes expansion and prevents apoptosis of cumulus cells in vitro.

Cumulus expansion is essential for ovulation and oocyte maturation in mammals. Previous studies suggest that this process requires certain cumulus expansion enabling factors, induced by LH surge, that activate SMAD signaling locally. However, their identities remain uncertain. Using a superovulated rat model, we showed that Bmp8 transcripts were abundant in cumulus cell-oocyte complexes (COCs) and their levels can be further induced during ovulation. By analyzing human COC-related transcriptomic datasets, BMP8 transcripts in cumulus cells were also found to be significantly elevated along with the maturation status and developmental competence of enclosed oocytes. In cultured rat COCs, treatment with recombinant BMP8A protein activated both SMAD1/5/8 and SMAD2/3 pathways; the resulting SMAD2/3 signaling induced COC expansion as well as the expression of COC expansion-related genes, whereas the resulting SMAD2/3 and SMAD1/5/8 activations were both required for protecting expanded cumulus cells from apoptosis. Taken together, our data demonstrated that addition of BMP8 protein in the in vitro rat COC cultures not only promotes cumulus expansion but also sustains survival of expanded cumulus cells via different SMAD downstreams. With these capabilities, BMP8 may have clinical applications to ameliorate the fertilizability and subsequent developmental competence of the enclosed oocytes when doing in vitro COC maturation.

Human BMP8A suppresses luteinization of rat granulosa cells via the SMAD1/5/8 pathway.

Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome. The correlation between the BMP8 levels in granulosa cells and the decline in ovarian function in these subjects suggests that BMP8 protein may be involved in the regulation of granulosa cell function(s). Using a rat model, we demonstrated that human BMP8A protein activates the SMAD1/5/8 and the SMAD2/3 pathways simultaneously in both immature and mature granulosa cells. Furthermore, the expression of potential type I and type II receptors used by BMP8 in rat granulosa cells was characterized. We found that BMP8A treatment can significantly inhibit gonadotropin-induced progesterone production and steroidogenesis-related gene expression in granulosa cells. Pathway dissection using receptor inhibitors further revealed that such inhibitory effects occur specifically through the BMP8-activated SMAD1/5/8, but not SMAD2/3, pathway. Taken together, considering its abundance and possible functions in granulosa cells, we suggest that BMP8 may act as a novel luteinization inhibitor in growing follicles.

In vivo selection reveals autophagy promotes adaptation of metastatic ovarian cancer cells to abdominal microenvironment.

Peritoneal dissemination is the most frequent metastatic route of ovarian cancer. However, due to the high heterogeneity in ovarian cancer, most conventional studies lack parental tumor controls relevant to metastases and, thus, it is difficult to trace the molecular changes of cancer cells along with the selection by the abdominal microenvironment. Here, we established an in vivo mouse peritoneal dissemination scheme that allowed us to select more aggressive sublines from parental ovarian cancer cells, including A2780 and SKOV-3. Microarray and gene profiling analyses indicated that autophagy-related genes were enriched in selected malignant sublines. Detection of LC3-II, p62 and autophagic puncta demonstrated that these malignant variants were more sensitive to autophagic induction when exposed to diverse stress conditions, such as high cell density, starvation and drug treatment. As compared with parental A2780, the selected variant acquired the ability to grow better under high-density stress; however, this effect was reversed by addition of autophagic inhibitors or knockdown of ATG5. When analyzing the clinical profiles of autophagy-related genes identified to be enriched in malignant A2780 variant, 73% of them had prognostic significance for the survival of ovarian cancer patients. Taken together, our findings indicate that an increase in autophagic potency among ovarian cancer cells is crucial for selection of metastatic colonies in the abdominal microenvironment. In addition, the derived autophagic gene profile can not only predict prognosis well but can also be potentially applied to precision medicine for identifying those ovarian cancer patients suitable for taking anti-autophagy cancer drugs.

BMP8A sustains spermatogenesis by activating both SMAD1/5/8 and SMAD2/3 in spermatogonia.

Mutation in either of the genes encoding bone morphogenetic protein (BMP) 8A or 8B (Bmp8a or Bmp8b) causes postnatal depletion of spermatogonia in mice. We found that Bmp8a, but not Bmp8b, was expressed predominantly in the neonatal mouse spermatogonia. Although most BMPs induce activation of SMADs 1, 5, and 8 (SMAD1/5/8), but not SMADs 2 and 3 (SMAD2/3), we found that BMP8A induced signaling through both sets of transcription factors. In undifferentiated mouse spermatogonia, BMP8A activated SMAD1/5/8 through receptor complexes formed by ALK3 and either ACVR2A or BMPR2 and activated SMAD2/3 through receptor complexes formed by ALK5 and ACVR2A, ACVR2B, or TGFBR2. Signaling through SMAD2/3 promoted the proliferation of germ cells, whereas that through SMAD1/5/8 directed the subsequent differentiation of spermatogonia. BMP8A promoted spermatogenesis in cultured mouse testis explants, and the resulting spermatids were functionally competent for fertilization. These results suggest that the dual role of BMP8A in promoting proliferation and differentiation of spermatogonia may be exploited clinically to treat male infertility.

Thyrostimulin-TSHR signaling promotes the proliferation of NIH:OVCAR-3 ovarian cancer cells via trans-regulation of the EGFR pathway.

Gonadotropin signaling plays an indispensable role in ovarian cancer progression. We previously have demonstrated that thyrostimulin and thyroid-stimulating hormone receptor (TSHR), the most ancient glycoprotein hormone and receptor pair that evolved much earlier than the gonadotropin systems, co-exist in the ovary. However, whether thyrostimulin-driven TSHR activation contributes to ovarian cancer progression in a similar way to gonadotropin receptors has never been explored. In this study, we first found that TSHR is expressed in both rat normal ovarian surface epithelium and human epithelial ovarian cancers (EOCs). Using human NIH:OVCAR-3 as a cell model, we demonstrated that thyrostimulin promotes EOC cell proliferation as strongly as gonadotropins. Thyrostimulin treatment not only activated adenylyl cyclase and the subsequent PKA, MEK-ERK1/2 and PI3K-AKT signal cascades, but also trans-activated EGFR signaling. Signaling dissection using diverse inhibitors indicated that EOC cell proliferation driven by thyrostimulin-TSHR signaling is PKA independent, but does require the involvement of the MEK-ERK and PI3K-AKT signal cascades, which are activated mainly via the trans-activation of EGFR. Thus, not only have we proved that this ancient glycoprotein hormone system is involved in NIH:OVCAR-3 cell proliferation for the first time, but also that it may possibly become a novel oncotarget when studying ovarian cancer.

NMU signaling promotes endometrial cancer cell progression by modulating adhesion signaling.

Neuromedin U (NMU) was originally named based on its strong uterine contractile activity, but little is known regarding its signaling/functions in utero. We identified that NMU and one of its receptors, NMUR2, are not only present in normal uterine endometrium but also co-expressed in endometrial cancer tissues, where the NMU level is correlated with the malignant grades and survival of patients. Cell-based assays further confirmed that NMU signaling can promote cell motility and proliferation of endometrial cancer cells derived from grade II tumors. Activation of NMU pathway in these endometrial cancer cells is required in order to sustain expression of various adhesion molecules, such as CD44 and integrin alpha1, as well as production of their corresponding extracellular matrix ligands, hyaluronan and collagen IV; it also increased the activity of SRC and its downstream proteins RHOA and RAC1. Thus, it is concluded that NMU pathway positively controls the adhesion signaling-SRC-Rho GTPase axis in the tested endometrial cancer cells and that changes in cell motility and proliferation can occur when there is manipulation of NMU signaling in these cells either in vitro or in vivo. Intriguingly, this novel mechanism also explains how NMU signaling promotes the EGFR-driven and TGFß receptor-driven mesenchymal transitions. Through the above axis, NMU signaling not only can promote malignancy of the tested endometrial cancer cells directly, but also helps these cells to become more sensitive to niche growth factors in their microenvironment.

Identifying a Neuromedin U Receptor 2 Splice Variant and Determining Its Roles in the Regulation of Signaling and Tumorigenesis In Vitro.

Neuromedin U (NMU) activates two G protein-coupled receptors, NMUR1 and NMUR2; this signaling not only controls many physiological responses but also promotes tumorigenesis in diverse tissues. We recently identified a novel truncated NMUR2 derived by alternative splicing, namely NMUR2S, from human ovarian cancer cDNA. Sequence analysis, cell surface ELISA and immunocytochemical staining using 293T cells indicated that NMUR2S can be expressed well on the cell surface as a six-transmembrane protein. Receptor pull-down and fluorescent resonance energy transfer assays demonstrated that NMUR1, NMUR2 and this newly discovered NMUR2S can not only form homomeric complexes but also heteromeric complexes with each other. Although not activated by NMU itself, functional assay in combination with receptor quantification and radio-ligand binding in 293T cells indicated that NMUR2S does not alter the translocation and stability of NMUR1 or NMUR2, but rather effectively dampens their signaling by blocking their NMU binding capability through receptor heterodimerization. We further demonstrated that NMU signaling is significantly up-regulated in human ovarian cancers, whereas expression of NMUR2S can block endogenous NMU signaling and further lead to suppression of proliferation in SKOV-3 ovarian cancer cells. In contrast, in monocytic THP-1 cells that express comparable levels of NMUR1 and NMUR2S, depletion of NMUR2S restored both the signaling and effect of NMU. Thus, these results not only reveal the presence of previously uncharacterized heteromeric relationships among NMU receptors but also provide NMUR2S as a potential therapeutic target for the future treatment of NMU signaling-mediated cancers.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

Multi-functional norrin is a ligand for the LGR4 receptor.

Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

Ovarian regulation of neuromedin U and its local actions in the ovary, mediated through neuromedin U receptor 2.

Neuromedin U (NMU) was originally identified as an anorexigenic peptide that modulates appetite as well as energy homeostasis through the brain-gut axis. Although growing evidence has linked NMU activity with the development of female reproductive organs, no direct expression of and function for NMU in these organs has been pinpointed. Using a superovulated rat model, we found that NMU is directly expressed in the ovary, where its transcript level is tightly regulated by gonadotropins. Ovarian microdissection and immunohistochemical staining showed clearly that NMU is expressed mainly in theca/interstitial cells and to a moderate extent in granulosa cells. Primary cell studies together with reporter assays indicated the Nmu mRNA level in these cells is strongly induced via cAMP signaling, whereas this increase in expression can be reversed by the degradation message residing within its 3'-untranslated region, which recruits cis-acting mRNA degradation mechanisms, such as the gonadotropin-induced zinc finger RNA-binding protein Zfp36l1. This study also demonstrated that NMUR2, but not NMUR1, is the dominant NMU receptor in the ovary, where its expression is restricted to theca/interstitial cells. Treatment with NMU led to induction of the early response c-Fos gene, phosphorylation of extracellular signal-regulated kinase 1/2, and promotion of progesterone production in both developing and mature theca/interstitial cells. Taken as a whole, this study demonstrates that NMU and NMU receptor 2 compose a novel autocrine system in theca/interstitial cells in which the intensity of signaling is tightly controlled by gonadotropins.

CXCL17, an orphan chemokine, acts as a novel angiogenic and anti-inflammatory factor.

Chemokines play pivotal roles in the recruitment of various immune cells to diverse tissues in both physiological and pathological conditions. CXCL17 is an orphan chemokine preliminarily found to be involved in tumor angiogenesis. However, its protein nature, as well as its endogenous bioactivity, has not been well clarified. Using real-time PCR, immunohistochemical staining, and Western blotting, we found that CXCL17 is highly expressed in both a constitutive and inducible manner in the rat gastric mucosa, where it undergoes endoproteolysis during protein maturation. The mature CXCL17 exhibited strong chemoattractant abilities targeting monocytes and macrophages, potentially through ERK1/2 and p38 but not JNK signaling. CXCL17 also induced the production of proangiogenic factors such as vascular endothelial growth factor A from treated monocytes. Furthermore, in contrast to other CXC chemokines that accelerate inflammatory responses, CXCL17 showed novel anti-inflammatory effects on LPS-activated macrophages. Therefore, our data suggest that CXCL17 in the gastric lamina propria may play an important role in tissue repair and anti-inflammation, both of which help to maintain the integrity of the gastric mucosa.

DAN (NBL1) specifically antagonizes BMP2 and BMP4 and modulates the actions of GDF9, BMP2, and BMP4 in the rat ovary.

Although differential screening-selected gene aberrative in neuroblastoma (DAN, official symbol NBL1) is the founding member of the DAN subfamily of bone morphogenetic protein (BMP) antagonists, its antagonizing targets, gene regulation, and physiological functions remain unclear. Using diverse cell expression systems, we found that the generation of bioactive DAN is likely to be cell type specific. Unlike other phylogenetically close members, which are covalently linked homodimers, DAN forms a noncovalently linked homodimer during folding. Purified recombinant DAN specifically blocked signaling of BMP2 and BMP4 but not that of other ovarian-expressed transforming growth factor-beta members. Although widely distributed in many organs, DAN transcript level was periodically regulated by gonadotropins. Ovarian microdissection indicated that NBL1 (DAN) mRNA is mainly expressed in granulosa cells, where its transcript level is up-regulated by the gonadotropin-driven cAMP cascade. We further investigated the local regulation and ovarian functions of DAN. NBL1 (DAN) mRNA expression in granulosa cells was up-regulated by oocyte-derived growth differentiation factor 9 (GDF9), whereas treatment with DAN significantly reversed the inhibitory effect of BMP4 on follicle-stimulating hormone-induced progesterone production in cultured granulosa cells. Our findings suggest the DAN gradient in granulosa cells, established by oocyte-derived GDF9, may serve as an antagonist barrier that modulates the actions of theca-derived BMP4 and granulosa/theca-derived BMP2 during folliculogenesis both spatially and temporally.

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug.

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

Exclusive expression of a membrane-bound Spink3-interacting serine protease-like protein TESPL in mouse testis.

We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5.

Thyrostimulin, but not thyroid-stimulating hormone (TSH), acts as a paracrine regulator to activate the TSH receptor in mammalian ovary.

The thyroid-stimulating hormone receptor (TSHR), activated by either TSH or the newly discovered glycoprotein hormone thyrostimulin, plays a central role in the control of body metabolism. Interestingly, in addition to its thyroid expression, we discovered that the mRNA level of TSHR is periodically regulated in rat ovary by gonadotropins. Ovarian microdissection followed by real-time PCR analysis indicated that granulosa cells show the highest level of TSHR expression. Cultures of follicles and primary granulosa cells demonstrated that the level of TSHR is up-regulated and decreased by the gonadotropin-driven cAMP cascade and estradiol production, respectively. Furthermore, in contrast to the negligible expression of TSH in the ovary, we also found by real-time PCR and immunohistochemical analysis that thyrostimulin is expressed mainly in oocytes. Evolving before the appearance of gonadotropins, thyrostimulin is considered the most ancestral glycoprotein hormone. Therefore, the presence of thyrostimulin in the ovary suggests that it may have a primitive function in reproduction when it activates ovarian TSHR. Next, we generated recombinant thyrostimulin protein and characterized its non-covalent heterodimeric nature. Using purified recombinant thyrostimulin, we show that the human ovarian cell line NIH:OVCAR-3 also expresses endogenous and functional TSHR. Using cultured rat granulosa cells isolated from different ovarian stages, we found that treatments with thyrostimulin significantly increase cAMP production and the c-fos gene response in the presence of gonadotropins. Thus, this study demonstrates that oocyte-derived thyrostimulin and granulosa cell-expressed TSHR compose a novel paracrine system in the ovary, where the activity is tightly controlled by gonadotropins.

Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors.

G proteins, further divided into four subfamilies (G(s), G(q), G(12) and G(i)) based on their Galpha subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Galpha signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Galpha. Therefore, our aim was to design a chimeric reporting system that covers responses from all Galpha subfamilies simultaneously. Because G(s) activates cAMP response element (CRE)-driven genes whereas G(q) and G(12) activate serum response element (SRE)-driven genes, we therefore incorporated 2x CRE and 5x SRE (2CRE5SRE) into a promoter for driving luciferase expression. To further report G(i) signals, a 2CRE5SRE-driven chimeric G(qi), in which the C-terminus of G(q) is replaced by that of G(i), was integrated to switch the responses of G(i)-coupled GPCRs to the G(q) signaling. The novel reporter system showed a strong signal amplification when activated by neuromedin U receptor 1 (mainly activates G(q)), neuromedin U receptor 2 (mainly activates G(i)) or luteinizing hormone receptor (mainly through the G(s) and G(q) pathways). In addition, 293T cells stably carrying our reporter construct showed a similar sensitivity to the radioactive cAMP assay when revealing the constitutive signal from gain-of-function mutants of luteinizing hormone receptor. To our knowledge, this is the first reporting system capable of covering the G(s), G(q), G(12) and G(i) signals and revealing the phenomena of constitutively active GPCRs. Such a universal platform will benefit future high-throughput screening and drug designs for any GPCR.

Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus.

PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 A resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 A.