Gene expression profiling of Nm23-H2 overexpressing CAL 27 cells using DNA microarray

Nm23-H1/NDPKA and Nm23-H2/NDPKB belong to a large family of NDP kinases, group of structurally and functionally closely related enzymes. The Nm23/NDPs are known to catalyse the transfer of terminal phosphates from ATP to other NTPs and dNTPs. Besides their role in the maintenance of the cells NTP pool the nm23 genes/proteins are known to have additional different biological functions, the most important being its metastasis suppressor activity. The complete picture of roles, actions and targets of nm23 genes/proteins is yet to be discovered. Our goal was to identify the downstream targets of Nm23-H2 by subjecting Nm23-H2 overexpressing CAL 27 cells (oral squamous cell carcinoma of the tongue) to microarray analysis. Using this powerful technology we identified genes, groups of genes and signalling pathways that could be clustered into several groups: apoptosis related genes, cell cycle and DNA damage, TGFbeta (transforming growth factor beta) signalling pathway and related molecules, WNT signalling pathway, differentiation and epithelial structural and related molecules, cell adhesion, metalloproteinases and their inhibitors, vesicular transport related molecules, proteasome associated, ubiquitin mediated proteolysis and several metabolic pathways. Based on these results we suggest that nm23-H2 might have an important role in oral squamous cell carcinoma which is to be confirmed by future studies. Key words: nm23-H2, DNA microarray, oral squamous cell carcinoma.     

Nm23-H1 expression in intrahepatic or extrahepatic metastases of hepatocellular carcinoma

Abstract: Aims/Background: Decreased expression of nm23, a putative metastasis suppressor gene, has been reported to be related to either intrahepatic metastasis or a poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to elucidate the true role of nm23-H1 expression in both intrahepatic and distant metastases of HCC. Methods: Thirteen patients with single-nodule HCC, seven patients with HCC having satellite nodules and seven patients with HCCs having extrahepatic metastases were included in this study. The expression of nm23-H1 protein was immunohistochemically examined in both primary and metastatic nodules. Results: Ten of 13 single-nodule HCCs were found to overexpress nm23-H1 protein. All main tumors, having satellite nodules, were found to overexpress nm23-H1 protein, except for two HCCs, which only partially expressed nm23-H1 protein. Regarding the nm23-H1 expression in intrahepatic metastases, most nodules overexpressed the protein. The expression of nm23-H1 was found to be low in only one intrahepatic metastasis specimen, while its primary tumor was also found to show a low expression of nm23-H1 protein. Microscopic portal vein invasion was found in three of the five patients studied, and all cancer cells in portal invasion overexpressed nm23-H1 protein. Nm23-H1 protein was expressed in all distant metastatic tumors and the staining intensity of most metastatic nodules was similar to that of the primary tumors. Conclusions: Our study demonstrated that nm23-H1 expression did not always decrease but instead tended to increase at both intrahepatic and extrahepatic metastatic sites. Based on these findings, nm23-H1 expression is not considered to be a reliable indicator of either intrahepatic or distant metastasis in HCC.     

Loss of heterozygosity of thenm23-H1 gene in human renal cell carcinomas

This study evaluates the potential contribution of thenm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for thenm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of thenm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at thenm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems thatnm23-H1 does not play a key role in the invasiveness of this tumor type.Abbreviations PCR polymerase chain reaction - LOH loss of heterozygosity     

Assessment of insulin sensitivity (S I) and glucose effectiveness (S G) from a standardized hyperglucidic breakfast test in type 2 diabetics exhibiting various levels of insulin resistance

We investigated the measurement of insulin sensitivity (S _I) with a standardized hyperglucidic breakfast (SHB) compared to minimal model analysis of an intravenous glucose tolerance test (S _I-IVGTT) in 17 patients clinically referred as type 2 diabetics, not yet treated by insulin, and representing a wide range of body mass index and S _I. To classify the patients, ten meal-tolerance test-based calculations of S _I (MTT-S _I) were compared to S _I-IVGTT, and their reference values and distribution were measured on a separate sample of 200 control SHBs and 209 control IVGTTs. Eight MTT-S_I indices exhibit significant correlations with S _I-IVGTT: Mari’s OGIS index, BIGTT-SI|_0–30–120, BIGTT-SI|_{0–60–120,} 1/G _b I _m, Caumo’s oral minimal model (OMM), Sluiter’s index “A” = 10⁴/(I _p·G _p), Matsuda’s composite index given by the formula ISIcomp = 10⁴/(I _b G _b I _m G _m)^0.5, S _I = 1/I _b G _b I _m G _m with r ² ranging between 0,53 and 0,28. S _I-IVGTT and S _I-MTT exhibited in the lower range a very different (non-normal) pattern of distribution and thus the cutoff value for defining insulin resistance varied among indices. With such cutoffs, S _I-MTT < 6.3 min^?1/(μU/ml) 10^?4 with Caumo’s OMM was the best predictor of insulin resistance defined as S _I-IVGTT < 2 min^?1/(μU/ml) 10^?4. Other indices, including OGIS and BIGTT, resulted in more misclassifications of patients. HOMA-IR and QUICKI were poor predictors. The formula $ S_{\rm G}=2.921\,{\text{e}}^{-0.185({\text{G}}_{60}-{\text{G}}_{0})}$ satisfactorily predicts IVGTT-derived glucose effectiveness in type 2 diabetics. Thus, SHB appears suitable for the measurement of S _I and S _G in type 2 diabetics, and the OMM seems to provide the most accurate SHB-derived index in this population.     

Interaction of the S phase regulator Cdc18 with cyclin-dependent kinase in fission yeast

The fission yeast gene cdc18⁺ is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G₁/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2⁺ gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G₁/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.     

Artin relation for smooth representations

Let G be a finite group. If G acts smoothly on a closed homotopy sphere S, we call S a smooth representation of G. The main result is: There is a function h_G such that for every smooth representation S of G, dimension S^G = h_G{dimension S^H|H proper subgroup of G} if and only if G has prime power order and G is not cyclic. In other words, only for a noncyclic p-group G is dimension S^G a universal function of the dimensions of the fixed sets S^H as H ranges over proper subgroups of G. This result is compared with an old theorem of Artin's dealing with dimensions of fixed sets of orthogonal representations of G.     

Inhibition of S1P Receptor 2 Attenuates Endothelial Dysfunction and Inhibits Atherogenesis in Apolipoprotein E-Deficient Mice

Aim: The bioactive lipid, sphingosine-1-phosphate (S1P), has various roles in the physiology and pathophysiology of many diseases. There are five S1P receptors; however, the role of each S1P receptor in atherogenesis is still obscure. Here we investigated the contribution of S1P receptor 2 (S1P2) to atherogenesis by using a specific S1P2 antagonist, ONO-5430514, in apolipoprotein E-deficient ( Apoe ^−/− ) mice. Methods: Apoe ^−/− mice fed with a western-type diet (WTD) received ONO-5430514 (30 mg/kg/day) or vehicle. To examine the effect on atherogenesis, Sudan IV staining, histological analysis, qPCR, and vascular reactivity assay was performed. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. Results: WTD-fed Apoe ^−/− mice had significantly higher S1P2 expression in the aorta compared with wild-type mice. S1P2 antagonist treatment for 20 weeks reduced atherosclerotic lesion development ( p ＜0.05). S1P2 antagonist treatment for 8 weeks ameliorated endothelial dysfunction ( p ＜0.05) accompanied with significant reduction of lipid deposition, macrophage accumulation, and inflammatory molecule expression in the aorta compared with vehicle. S1P2 antagonist attenuated the phosphorylation of JNK in the abdominal aorta compared with vehicle ( p ＜0.05). In HUVEC, S1P promoted inflammatory molecule expression such as MCP-1 and VCAM-1 ( p ＜0.001), which was attenuated by S1P2 antagonist or a JNK inhibitor ( p ＜0.01). S1P2 antagonist also inhibited S1P-induced JNK phosphorylation in HUVEC ( p ＜0.05). Conclusions: Our results suggested that an S1P2 antagonist attenuates endothelial dysfunction and prevents atherogenesis. S1P2, which promotes inflammatory activation of endothelial cells, might be a therapeutic target for atherosclerosis.     

Expression of nm23-H1 predicts lymph node involvement in colorectal carcinoma

PURPOSE: Reduced expression of the metastasis suppressor gene nm23-H1 has previously been correlated with high tumor metastatic potential and fatal clinical outcome in some tumors (e.g.,breast). For colorectal carcinomas, the findings are equivocal. METHODS: We have used a monoclonal antibody against nm23-H1 to investigate the expression in colorectal carcinomas at the time of primary curative surgery (RO resection) to assess if there was any relation between nm23-H1 expression and stage or histologic grade at the time of primary tumor removal. RESULTS: Of 100 colorectal carcinomas studied (Stages I, II, and III according UICC, all resected curatively), nm23-H1 immunoreactivity was weak in 41 (41 percent), moderate in 24 (24 percent), and strong in 35 (35 percent) cases. The grade of positivity against nm23-H1 was significantly lower in advanced stages of the disease (Stages II or III) (P < 0.001, chi-squared=52.8). In tumors with low or weak immunoreactivity against nm23-H1, frequency of lymph node metastases was significantly higher compared with those with moderate or strong staining (P < 0.001; chi-squared=50.58). Therefore, with a sensitivity of 93 percent and a specificity of 58 percent, low nm23-H1 immunoreactivity of the primary tumor, assessed at the time of surgery, is an indicator of the presence of lymph node metastases. CONCLUSIONS: Immunohisto-chemical evaluation of nm23-H1 in the primary tumor or in a biopsy is a useful predictor of stage of disease and presence of lymph node metastases in colorectal carcinomas and may have clinical significance,e.g.,in predicting optimal therapeutic regimes.     

B7-H1 (PD-L1) on T cells is required for T-cell-mediated conditioning of dendritic cell maturation

Studies have shown that T-cell-dendritic cell (DC) interaction is required for efficient DC maturation. However, the identities of the molecules that mediate the interaction in vivo are largely unknown. Here, we show that maturation of DCs as well as CD8 T-cell responses were impaired in B7-H1-deficient (B7-H1^−/−) mice to influenza virus infection. Both defects were restored by transferring B7-H1-expressing naïve T cells into B7-H1^−/− mice. Similarly, transferring DCs from wild-type mice or from RAG1^−/− mice that had been injected with B7-H1-expressing naïve T cells also restored CD8 T-cell responses in B7-H1^−/− mice. These results demonstrate that B7-H1 on naïve T cells is required to condition immature DCs to undergo efficient maturation when they encounter microbial infection. In return, the mature DCs stimulate a robust T-cell reponse against the infecting pathogen.     

Molecular basis of plant-specific acid activation of K+ uptake channels

During stomatal opening potassium uptake into guard cells and K⁺ channel activation is tightly coupled to proton extrusion. The pH sensor of the K⁺ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K⁺ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K⁺ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K⁺ uptake channels.     

A study on nm23-H1 expression in diffuse large B-cell lymphoma that was treated with CyclOBEAP plus rituximab therapy

In our previous study on nm23-H1 expression with diffuse large B-cell lymphoma (DLBCL), we found that patients with positive nm23-H1 had significantly poorer prognosis than patients with negative nm23-H1. We examined whether nm23-H1 is a prognostic factor of DLBCL in the rituximab era. The subjects were 101 DLBCL patients who underwent R-CyclOBEAP (rituximab, cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, and prednisolone) therapy and in whom markers could be analyzed. We evaluated CD5, CD10, BCL2, BCL6, MUM1, and nm23-H1 expression by immunohistochemistry. Ninety-four DLBCL patients who underwent CyclOBEAP therapy were assumed as historical controls. Among DLBCL patients who underwent CyclOBEAP therapy, BCL2 positivity, MUM1 positivity, non-germinal center B-cell (non-GCB), and nm23-H1 positivity were associated with significantly shorter overall survival (OS) and progression-free survival (PFS). On the other hand, among DLBCL patients who underwent R-CyclOBEAP therapy, the 5-year OS rates of the nm23-H1-positive DLBCL (n = 32) and nm23-H1-negative DLBCL groups (n = 69) were 65% and 97%, respectively (p = 0.001), with 5-year PFS rates of 51% and 89%, respectively (p = 0.001). In the rituximab era, BCL2, MUM1, and non-GCB were not prognostic factors. We demonstrated that among patients with DLBCL who underwent R-CyclOBEAP therapy, patients with nm23-H1 expression had a significantly poorer prognosis than patients without nm23-H1 expression. These results suggest an important role for nm23-H1 in malignant progression and a potential therapeutic target for DLBCL.     

Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn²⁺ for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.     

Enzyme-to-enzyme channelling of symmetric Krebs cycle intermediates in pancreatic islet cells

Summary Tumoural islet cells of the RINm5F line were incubated for 120 min in the presence of [2-¹³C]propionate (10 mmol/l), and the ¹³C enrichment of lactate released in the incubation medium was monitored by ¹³C nuclear magnetic resonance. The C₃/C₂ ratio of resonance areas was much lower than that found with naturally ¹³C-enriched lactate. This reveals that symmetric Krebs cycle intermediates undergo oriented transfer in the sequence of reactions catalysed by succinate thiokinase, succinate dehydrogenate and fumarase in the mitochondria of islet cells.Abbreviations NMR nuclear magnetic resonance     

nm23-H1 immunoreactivity as a prognostic factor in differentiated thyroid carcinoma

Several prognostic factors have been proposed to identify the patients at risk to develop metastases in differentiated thyroid carcinoma. Reduced nm23-H1 expression (a metastatic suppressor gene) has been correlated with high tumor metastatic potential in various human carcinomas, but the results obtained in differentiated thyroid carcinoma remain controversial. To elucidate the usefulness of nm23-H1 as a differentiated thyroid carcinoma prognosis factor, we evaluate the relationship between nm23-H1 immunoreactivity as well as both clinical status and patient outcome. For this purpose, thyroid resected specimens obtained from 94 differentiated thyroid carcinoma consecutive patients (64 papillary and 30 follicular) with at least 5 yr of follow-up were stained using monoclonal antibody to nm23-H1. We did not observe any relationship between nm23-H1 immunoreactivity and age, gender, initial differentiated thyroid carcinoma stage, local recurrence, or distant metastases in patients with papillary carcinoma. However, in patients with follicular carcinoma, a significant inverse association between metastatic disease and the expression of nm23-H1 product was obtained (P < 0.05). In addition, significant differences were found in the survival curves according to nm23-H1 immunoreactivity (log-rank P < 0.01). Finally, nm-23-H1 immunoreactivity was more specific but less sensitive than AMES score to predict metastases. In conclusion, our results suggest that nm23-H1 immunostaining could be added to the classic prognostic factors currently used to predict the outcome of patients with follicular thyroid carcinoma.     

Dynamic regulation of FGF23 by Fam20C phosphorylation,GalNAc-T3 glycosylation,and furin proteolysis

The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser¹⁸⁰, within the FGF23 R¹⁷⁶XXR¹⁷⁹/S¹⁸⁰AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.Protein kinases are evolutionarily conserved enzymes that regulate numerous cellular processes by transferring a molecule of phosphate from ATP to target substrates (1, 2). The vast majority of theses enzymes function within the nucleus and cytosol. In contrast, there are several examples of phosphorylated proteins that are secreted from the cell, which raises the question: What are the kinases that phosphorylate these secreted phosphoproteins? We recently identified a small family of secretory pathway kinases that phosphorylate secreted proteins and proteoglycans (3). These enzymes have N-terminal signal sequences that direct them to the lumen of the endoplasmic reticulum, where they encounter the proteins or proteoglycans that they phosphorylate. One member of this atypical kinase family is the family with sequence similarity 20, member C (Fam20C), which phosphorylates secreted proteins on Ser(S)-x-Glu(E)/pSer(pS) (S-x-E/pS) motifs (3, 4). Because Fam20C localizes within the secretory pathway and the vast majority of secreted phosphoproteins are phosphorylated on S-x-E/pS motifs, Fam20C has been proposed to play a major role in the generation of the secreted phosphoproteome (5–7). For example, ∼75% of human serum and cerebrospinal fluid phosphoproteins are phosphorylated on S-x-E/pS motifs (8, 9). This includes proteins important for tooth and bone formation, as well as numerous hormones. In most cases, the functional importance of these phosphorylation events is unknown.Loss-of-function mutations in the human FAM20C gene cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia (10, 11). Most Raine patients die within the first few weeks of life. Nonlethal cases have also been reported, and these patients develop hypophosphatemia as a result of elevated levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) (12). Additionally, Fam20C knockout (KO) mice develop renal phosphate wasting due to an increase in circulating bioactive FGF23 as well as severe hypophosphatemic rickets (13, 14). Thus, Fam20C has been proposed to be a regulator of FGF23; however, the molecular mechanisms underlying the control of this hormone by Fam20C are unclear.FGF23 is secreted from osteoblasts and osteocytes, and targets the kidney to regulate the reabsorption of phosphate and catabolism of 1,25-dihydroxyvitamin D₃ (15, 16). FGF23 inhibits renal phosphate transport by activating FGF receptors (FGFRs) in a manner that requires binding the coreceptor α-klotho (α-KL) (17). Binding of FGF23 to FGFRs/α-KL induces urinary excretion of phosphate by decreasing the abundance of the type II sodium-dependent phosphate cotransporters NPT2a and NPT2c (15, 16). Several genetic disorders of renal phosphate wasting are associated with alterations in FGF23 (18). X-linked hypophosphatemia is the most prevalent form of hypophosphatemic rickets (1:20,000), and is caused by loss-of-function mutations in the gene encoding the phosphate-regulating endopeptidase homolog X-linked, which is associated with elevated FGF23 expression (19, 20). A similar situation exists in patients with an autosomal recessive form of hypophosphatemic rickets caused by mutations in the ectonucleotide pyrophosphatase ENPP1 (21) and the Fam20C substrate dentin matrix protein 1 (DMP1) (22). These disorders share the common denominator of elevated FGF23.Other Mendelian disorders of renal phosphate handling have provided important insight into the regulation of FGF23 protein processing. FGF23 is inactivated in the Golgi by proteolysis within a highly conserved subtilisin-like proprotein convertase (SPC) site, ¹⁷⁶RHTR¹⁷⁹/S¹⁸⁰AE¹⁸², generating inactive N- and C-terminal fragments (23). Gain-of-function missense mutations in FGF23 cause autosomal dominant hypophosphatemic rickets (ADHR) (24). These mutations substitute the Arg (R) residues within the SPC cleavage site and render the protein resistant to proteolysis (25). Conversely, loss-of-function mutations in FGF23 cause familial tumoral calcinosis (FTC), a hyperphosphatemic disorder characterized by often severe ectopic and vascular calcifications (26). FTC is also caused by loss-of-function mutations in polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), a glycosyltransferase that O-glycosylates FGF23 at Thr¹⁷⁸ within the SPC cleavage site (27, 28). These inactivating GalNAc-T3 mutations prevent O-glycosylation of FGF23, which produces a hormone more susceptible to SPC proteolysis (28, 29). Collectively, these studies suggest that FGF23 processing is a highly regulated and physiologically important process.Here we demonstrate that Fam20C regulates FGF23 by phosphorylation of Ser¹⁸⁰, a residue that neighbors the SPC site (R¹⁷⁶H¹⁷⁷T¹⁷⁸R¹⁷⁹/S¹⁸⁰). We show that Ser¹⁸⁰ phosphorylation inhibits GalNAc-T3 O-glycosylation of Thr¹⁷⁸, thereby promoting furin (PCSK3)-dependent FGF23 proteolysis. Our results provide a plausible molecular mechanism by which loss of Fam20C leads to elevated levels of intact, biologically active FGF23 and subsequent hypophosphatemia. Furthermore, our results suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are dynamically regulated.     

The chemistry of the S-nitrosoglutathione/glutathione system

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems—e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1–10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with ¹⁵N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N₂O₃), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO + GSH.