GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

Highly conserved protein Rv1211 in Mycobacterium tuberculosis is a natively unfolded protein that binds to a calmodulin antagonist, trifluoperazine.

Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and performed structural and biochemical studies of Rv1211 and its interaction with Ca2+ or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2+. Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of 15N-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 µM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the suggestion of Rv1211 as a Ca2+-binding calmodulin-like protein, and show that Rv1211 is a natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness" in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins.

A Serum Marker for Early Pancreatic Cancer With a Possible Link to Diabetes.

BACKGROUND: Pancreatic cancer (PC) has a grim prognosis, and an early diagnostic biomarker has been highly desired. The molecular link between diabetes and PC has not been well established. METHODS: Bioinformatics screening was performed for a serum PC marker. Experiments in cell lines (5 PC and 1 normal cell lines), mouse models, and human tissue staining (37 PC and 10 normal cases) were performed to test asprosin production from PC. Asprosin's diagnostic performance was tested with serums from multi-center cohorts (347 PC, 209 normal, and 55 additional diabetic patients) and evaluated according to PC status, stages, and diabetic status, which was compared with that of CA19-9. RESULTS: Asprosin, a diabetes-related hormone, was found from the bioinformatics screening, and its production from PC was confirmed. Serum asprosin levels from multi-center cohorts yielded an age-adjusted diagnostic area under the curve (AUC) of 0.987 (95% confidence interval [CI] = 0.961 to 0.997), superior to that of CA19-9 (AUC = 0.876, 95% CI = 0.847 to 0.905), and a cut-off of 7.18 ng/mL, at which the validation set exhibited a sensitivity of 0.957 and a specificity of 0.924. Importantly, the performance was maintained in early-stage and non-metastatic PC, consistent with the tissue staining. A slightly lower performance against additional diabetic patients (n = 55) was restored by combining asprosin and CA19-9 (AUC = 0.985, 95% CI = 0.975 to 0.995). CONCLUSIONS: Asprosin is presented as an early-stage PC serum marker that may provide clues for PC-induced diabetes. Larger prospective clinical studies are warranted to solidify its utility.

Interaction between IDH1 WT and calmodulin and its implications for glioblastoma cell growth and migration.

Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.

Artemisia Capillaris leaves inhibit cell proliferation and induce apoptosis in hepatocellular carcinoma.

BACKGROUND: Natural product is one of the most important sources of drugs used in pharmaceutical therapeutics. Artemisia capillaris has been traditionally used as a hepatoprotective and anti-inflammatory agent. In this study, we extracted an ethanol fraction (LAC117) from the dried leaves of Artemisia capillaris and identified its anticancer activity and mechanism of action against hepatocellular carcinoma (HCC). METHODS: Anti-proliferative effect of LAC117 was evaluated by MTT assay and BrdU assay. The apoptotic effect of LAC117 on the expression of cleaved PARP and cleaved caspase-3 was evaluated by Western blot and immunohistochemistry from in vivo mouse xenograft, respectively. RESULTS: We found that LAC117 strongly suppressed the growth and proliferation of human HCC cell lines (HepG2 and Huh7). Induction of apoptosis was evidenced by the increases of cleaved caspase-3 and PARP as well as TUNEL-positive cells. Additionally, the pro-apoptotic effect of LAC117 was observed by a decrease in the expression of the XIAP and an increase in cytochrome c releases via mitochondrial membrane potential. Moreover, it significantly inhibited PI3K/AKT pathway in HCC in vivo and in vitro. LAC117 suppressed tumor growth in an ex vivo model as well as in vivo mouse xenograft by inducing apoptosis and inhibiting tumor cell proliferation. CONCLUSIONS: The present study highlights that LAC117 could not only efficiently induce apoptosis, but also inhibit the growth of human HCC cells by blocking the PI3K/AKT signaling pathway, suggesting that LAC117 would be a potentially useful drug candidate against HCC.

Prediction of glycated hemoglobin levels at 3 months after metabolic surgery based on the 7-day plasma metabolic profile.

Metabolic surgery has been shown to provide better glycemic control for type 2 diabetes than conventional therapies. Still, the outcomes of the surgery are variable, and prognostic markers reflecting the metabolic changes by the surgery are yet to be established. NMR-based plasma metabolomics followed by multivariate regression was used to test the correlation between the metabolomic profile at 7-days after surgery and glycated hemoglobin (HbA1c) levels at 3-months (and up to 12 months with less patients), and to identify the relevant markers. Metabolomic profiles at 7-days could differentiate the patients according to the HbA1c improvement status at 3-months. The HbA1c values were predicted based on the metabolomics profile with partial least square regression, and found to be correlated with the observed values. Metabolite analysis suggested that 3-Hydroxybutyrate (3-HB) and glucose contributes to this prediction, and the [3-HB]/[glucose] exhibited a modest to good correlation with the HbA1c level at 3-months. The prediction of 3-month HbA1c using 7-day metabolomic profile and the suggested new criterion [3-HB]/[glucose] could augment current prognostic modalities and help clinicians decide if drug therapy is necessary.

Heteronuclear NMR as a 4-in-1 analytical platform for detecting modification-specific signatures of therapeutic insulin formulations.

Detecting possible modifications of therapeutic proteins is a critical element of the quality control of protein drugs. Typically, a number of techniques are used to evaluate different modifications of therapeutic protein formulations. Using heteronuclear NMR spectroscopy, we show that the difference between various insulin formulations can be detected "as is" with little pretreatment and quickly. As an application to the quality control of insulin formulations, the NMR approach was compared with four different analytical methods: with reverse phase high pressure liquid chromatography (HPLC) (for mutations), with size exclusion chromatography (for oligomerization), with electrophoresis (for denaturation), and with mass spectrometry (for deamidation). All of the results showed that this single NMR method can provide the specific signatures for each modification and information that is at least equivalent to that offered by the conventional analytical methods. Importantly, NMR could yield information at each amino acid residue level which no other technique provided. The suggested NMR method, then, can be considered to be a facile and effective means of evaluating therapeutic protein formulations in a multifaceted way.

Effects of peribulbar anesthesia (sub-Tenon injection of a local anesthetic) on akinesia of extraocular muscles, mydriasis, and intraoperative and postoperative analgesia in dogs undergoing phacoemulsification.

OBJECTIVE: To evaluate the effects of peribulbar anesthesia (sub-Tenon injection of lidocaine hydrochloride) on akinesia of extraocular muscles, mydriasis, and intraoperative and postoperative analgesia in dogs undergoing phacoemulsification. ANIMALS: 14 Beagles with ophthalmically normal eyes. PROCEDURES: A blinded randomized controlled trial was performed. Dogs were anesthetized and assigned to 2 treatments: concurrent sub-Tenon injection of 2% lidocaine hydrochloride solution (2 mL) and IV injection of saline (0.9% NaCl) solution (0.02 mL/kg; lidocaine group [n = 7]) or concurrent sub-Tenon injection of saline solution (2 mL) and IV injection of 0.2 mg of atracurium/kg (0.02 mL/kg; control group [7]). Pupils were dilated by topical application of a combined tropicamide and phenylephrine ophthalmic solution. Ten minutes after the injections, pupil diameter was measured and phacoemulsification was performed. End-tidal isoflurane concentration was used to evaluate intraoperative pain. Subjective pain scores were recorded during the postoperative period. RESULTS: Akinesia was induced and maintained throughout the surgery in all eyes. Mean ± SD pupil diameter was significantly greater in the lidocaine group (13.7 ± 0.7 mm) than in the control group (12.2 ± 0.8 mm). Isoflurane requirements were significantly lower in the lidocaine group than the control group. However, postoperative pain scores were not significantly different between the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Sub-Tenon injection of lidocaine was an effective method for inducing akinesia of extraocular muscles, mydriasis, and intraoperative analgesia for phacoemulsification in dogs. Therefore, this could be another option for surgical field exposure and pain management during phacoemulsification in dogs.

Characterization of the binding sites for the interactions between FKBP12 and intracellular calcium release channels.

FKBP12, an FK506 binding protein, interacts with type 1 ryanodine receptor (RyR1) and modulates its calcium channel activity. However, there are many opposing reports of FKBP12's interaction with other related calcium channels, such as type 1 IP(3) receptor and type 3 ryanodine receptor (IP(3)R1 and RyR3). In addition, the involvement of the prolyl-dipeptide motif in the calcium channels and the corresponding binding residues in FKBP12 remain controversial. Through pulldown assays with recombinant proteins, we provide biochemical evidence of the interaction between FKBP12 and RyR1, RyR3 and IP(3)R1. Using NMR chemical shift mapping, we show that the important binding residues in FKBP12 are located in its hydrophobic FK506 binding region. Consistently, we demonstrate that FK506 can competitively inhibit the interaction between FKBP12 and the dipeptide motifs of the calcium channels. We believe our results shed lights on the binding mechanism of calcium channel-FKBP12 interaction.

Global dynamic conformational changes in the suppressor domain of IP3 receptor by stepwise binding of the two lobes of calmodulin.

The roles of calmodulin (CaM) have been key points of controversy in the regulation of inositol-1,4,5-trisphosphate receptor (IP(3)R). To address the issue, we studied the interaction between CaM and the suppressor domain of IP(3)R, a key allosteric regulatory domain. First, by means of a pulldown and a fluorescence titration experiment, we confirmed the interaction. Through subsequent NMR binding experiments, we observed dramatic peak disappearances of the suppressor domain on interaction with apo-CaM. The data indicated that apo-CaM induces large-scale dynamic conformational changes in the suppressor domain, involving partial unfolding and subdomain rearrangement. Analysis of the NMR data of CaM surprisingly revealed that its C lobe alone can cause such changes. Further binding experiments showed that calcium allows the free N lobe to bind to the suppressor domain, which induces extra conformational changes in both of the proteins. These results were also confirmed with CaM deletion mutants with either the N or C lobe. On the basis of this novel binding mechanism, we propose a model in which the partial unfolding of the suppressor domain by apo-CaM and the stepwise binding of the N lobe of CaM to the suppressor domain are important elements of calcium/CaM inhibition of IP(3)R. We believe that our working model encompasses previous regulation mechanisms of IP(3)R by calcium/CaM and provides new insights into the CaM-target interaction.

Predicting idiopathic toxicity of cisplatin by a pharmacometabonomic approach.

Cisplatin has been one of the most widely used anticancer agents, but its nephrotoxicity remains a dose-limiting complication. Here, we evaluated the idiopathic nature and the predose prediction of cisplatin-induced nephrotoxicity using a nuclear magnetic resonance (NMR)-based pharmacometabonomic approach. Cisplatin produced serious toxic responses in some animals (toxic group), but had little effect in others (nontoxic group), as judged by hematological and histological results. The individual metabolic profiles, assessed by urine NMR spectra, showed large differences between the post-administration profiles of the two groups, indicating the relevance of the NMR approach. Importantly, multivariate analysis of the NMR data showed that the toxic and nontoxic groups can be differentiated based on the pretreatment metabolite profiles. Leave-one-out analysis, performed to evaluate the practical performance of our approach, gave a 66% accuracy rate in predicting toxic responses based on the pretreatment metabolite profiles. Hence, we provide a working model that can explain the idiopathic toxicity mechanism based on marker metabolites found by NMR analysis consistent with tissue NADH measurements. Thus, a pharmacometabonomic approach using pretreatment metabolite profiles may help expedite personalized chemotherapy of anticancer drugs.

Differentiation of antlers from deer on different feeds using an NMR-based metabolomics approach.

Deer antler has been widely used as a dietary supplement for hundreds of years in Asian countries. The chemical composition of deer antlers strongly depends on the growth conditions of the deer, especially the feeds, but the effects of different feeds on deer antlers have not been studied. To expand our knowledge of the chemical constituents of deer antler and establish an efficient way of differentiating antlers obtained with different feeds, we applied an NMR-based metabolomics approach and OPLS-DA multivariate analysis. We show that the antlers from one species on two different feeds, made from grass or mulberry trees, can be reliably differentiated by our metabolomics approach. We identified chemical constituents of the deer antlers and the marker compounds that contribute to the difference between the feed groups. We also rigorously validated our differentiation approach by showing that it can correctly classify blind samples into their respective feed groups. Our approach is expected to help design feeds to produce antlers with more defined constituents, especially those with higher bioactivities.

A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer.

BACKGROUND & AIMS: Biliary tract cancer is highly lethal at presentation, with increasing mortality worldwide. Current diagnostic measures employing multiple criteria such as imaging, cytology, and serum tumor markers are not satisfactory, and a new diagnostic tool is needed. Because bile is a cognate metabolite-rich bio-fluid in the biliary ductal system, we tested a new metabolomic approach to develop an effective diagnostic tool. METHODS: Biles were collected prospectively from patients with cancer (n=17) or benign biliary tract diseases (n=21) with percutaneous or endoscopic methods. Nuclear magnetic resonance spectra (NMR) of these biles were analyzed using orthogonal partial least square discriminant analysis (OPLS-DA). RESULTS: The metabolomic 2-D score plot showed good separation between cancer and benign groups. The contributing NMR signals were analyzed using a statistical TOCSY approach. The diagnostic performance assessed by leave-one-out analysis exhibited 88% sensitivity and 81% specificity, better than the conventional markers (CEA, CA19-9, and bile cytology). CONCLUSION: The NMR-based metabolomics approach provides good performance in discriminating cancer and benign biliary duct diseases. The excellent predictability of the method suggests that it can, at least, augment the currently available diagnostic approaches.

Differentiation of cultivation sources of Ganoderma lucidum by NMR-based metabolomics approach.

INTRODUCTION: Ganoderma lucidum is a widely used and high-value medicinal natural product. Correct identification of its cultivation source is important for proper quality assurance, but is so far mostly dependent on subjective morphological examinations. OBJECTIVE: To develop an efficient way of discriminating the cultivation sources of Ganoderma lucidum, particularly those from Korea and China, the two major sources of the mushroom using NMR-based metabolomic differentiation. METHODOLOGY: Ganoderma lucidum samples were collected from Korea (26 samples) and China (20 samples), and their NMR spectra were obtained. The raw data were processed, and analysed using multivariate statistical analysis. RESULTS: Although conventional principal component analysis showed some overlaps, orthogonal projections to latent structure discriminant analysis (OPLS-DA) provided clean distinction between samples from the two countries. Contributing signals were also identified using S-plot, and further verified with independent t-test. Final validation of the model was obtained using prediction test of the unknowns, where the model predicted all of the 14 test samples correctly. Distinction between the cultivation sources within China was also established. CONCLUSION: The easiness and transferability of our NMR-based approach should contribute to addressing an important aspect of quality control process of Ganoderma lucidum. We believe the method can be easily applied to other herbal medical products.

Application of a 1H nuclear magnetic resonance (NMR) metabolomics approach combined with orthogonal projections to latent structure-discriminant analysis as an efficient tool for discriminating between Korean and Chinese herbal medicines.

Correct identification of the origins of herbal medical products is becoming increasingly important in tandem with the growing interest in alternative medicine. However, visual inspection of raw material is still the most widely used method, and newer scientific approaches are needed. To develop a more objective and efficient tool for discriminating herbal origins, particularly Korean and Chinese, we employed a nuclear magnetic resonance (NMR)-based metabolomics approach combined with an orthogonal projections to latent structure-discriminant analysis (OPLS-DA) multivariate analysis. We first analyzed the constituent metabolites of Scutellaria baicalensis through NMR studies. Subsequent holistic data analysis with OPLS-DA yielded a statistical model that could cleanly discriminate between the sample groups even in the presence of large structured noise. An analysis of the statistical total correlation spectroscopy (STOCSY) spectrum identified citric acid and arginine as the key discriminating metabolites for Korean and Chinese samples. As a validation of the discrimination model, we performed blind prediction tests of sample origins using an external test set. Our model correctly predicted the origins of all of the 11 test samples, demonstrating its robustness. We tested the wider applicability of the developed method with three additional herbal medicines from Korea and China and obtained very high prediction accuracy. The solid discriminatory power and statistical validity of our method suggest its general applicability for determining the origins of herbal medicines.

Effects of redox potential and Ca2+ on the inositol 1,4,5-trisphosphate receptor L3-1 loop region: implications for receptor regulation.

Inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major intracellular Ca(2+) channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP(3)R and the regulators, and whether it involves conformational changes of the lumenal regions of IP(3)R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca(2+), on the L3-1 lumenal loop region of IP(3)R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca(2+) showed little effect on the L3-1 conformation, suggesting that the regulation of Ca(2+) is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys(34) and Cys(42). A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys(34)-Cys(42) region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys(34) and Cys(42) may act together as a reduction sensor, and that Cys(65) may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca(2+) can regulate IP(3)R through totally different mechanisms: Ca(2+) by the indirect effect and the redox potential by direct action causing conformational changes.

Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling.

The SH3 domain is a versatile protein interaction motif that generally recognizes proline rich sequences (PRS). Recently, it has been shown that some SH3 domains in the endocytotic pathway can bind to ubiquitin. Moreover, Phe73 in the SH3 domain has been proposed to be an important determinant of the interaction, as the SH3 domains having Tyr73, either naturally or by mutation, failed to bind. Since SH3 domains are also important in immune receptor signaling, we investigated the interactions between immunologically relevant SH3 domains and ubiquitin. We observed that some of these SH3 domains can also bind to ubiquitin. Interestingly, we found that Nck2-SH3-3 bound to ubiquitin despite its Tyr at residue 73 (Tyr56 in our actual construct), but that CD2BP1-SH3 failed to bind, even though it has Phe at an equivalent position. Through detailed NMR binding studies on SH3 domains with Phes and Tyrs at the 73 position, we found that the two types of SH3 domains exhibit mechanistic differences in ubiquitin binding. We showed that the relative contribution of each binding sub-region in both SH3 domains and ubiquitin is quite different in the two binding modes. Such results raise the possibility that the mechanistic variety of these immunologically relevant SH3 domains might contribute to their functional diversity.

NMR-based metabolomics approach for the differentiation of ginseng (Panax ginseng) roots from different origins.

Agro-herbal materials vary in prices and qualities depending on the origin and age and the differentiation is both scientific and public health issue. Here, we describe a metabolomics approach used to discriminate ginseng roots from different sources. Six different types of ginseng roots from China and Korea were analyzed by NMR-based metabolomics. Chinese Dangsam showed prominent differences and was easily differentiated. The difference was mainly due to the large signals in the sugar region. We further analyzed the metabolomics results in subgroups. Jeonra (Korean), Choongcheong (Korean), and Chinese ginseng in subgroup 1 could be easily differentiated by the first two principal components. The loading plot for PC1 showed that the Jeonra and Chinese ginseng roots were mainly separated by sugar signals and methyl signals but that they were reverse-correlated. A diffusion-ordered spectroscopy (DOSY) analysis showed that the methyl signals are from high molecular weight compounds and that the sugar signals are either from oligosaccharides or ginsenosides. In subgroup 2, composed of Korean Choongcheong ginseng at different ages, we were able to see age-dependent transitions in the score plot. We believe our approach can be applied to detecting the adulteration of ginseng root powders and other herbal products from different origins.

Identification of residues participating in the interaction between an intraluminal loop of inositol 1,4,5-trisphosphate receptor and a conserved N-terminal region of chromogranin B.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a membrane channel that conducts calcium ions from the intracellular calcium stores. Despite a wealth of information on the cytoplasmic regulation of the IP3R, little is known about its regulation on the luminal side of the calcium stores. Here, we report studies on the IP3R intraluminal loop L3-2 and a conserved N-terminal region of chromogranin B. The IP3R loop is an important part of the channel's pore-forming region, and the chromogranin peptide has been shown to competitively inhibit calcium signaling by IP3R. Using the NMR titration approach, we showed that a part of the L3-2 is involved in a specific interaction with the chromogranin B peptide. Further NMR resonance assignments revealed that the 14th-20th residues of L3-2 are the keys to the binding to the chromogranin B peptide. Through detailed analysis of the data, we suggest a mechanism of IP3R regulation by chromogranin B involving conformational exchanges of the L3-2 region. Our report presents the findings of the first study on the interaction between the luminal loop of the IP3 receptor and its regulator at residue-resolution. The approaches described here should help to guide further studies on the interactions between the IP3R and other luminal side regulators.

Recombinant preparation and characterization of interactions for a calmodulin-binding chromogranin A peptide and calmodulin.

Chromogranin-derived peptides have important and varied biological activities. They affect a wide spectrum of targets such as fungal membranes, blood vessels, myocardial cells, and pancreatic cells. Despite the biological significance and the diverse activities, the molecular mechanisms of the interactions between the peptides and the target proteins have not been well understood. Here, we studied the interaction between a chromogranin A-derived peptide (CGA40-65) and its target protein, calmodulin, with NMR spectroscopy. Calmodulin was easily prepared with standard recombinant technology, but CGA40-65 posed challenges requiring multistep procedures. The recombinantly produced peptide retained the calmodulin-binding property of the full-length CGA, as shown by the HSQC binding experiment. By applying resonance assignments, we identified the residues in calmodulin involved in the CGA40-65 binding. We also found that the peak changes are close to those exhibited by the peptides having the wrap-around binding mechanism. Further analysis revealed that the CGA40-65-induced changes are more similar to those by CaMKIp peptide than those by smMLCKp peptide among the wrap-around binding peptides, suggesting that CGA40-65 can be categorized as a CaMKIp-like peptide. Our report is the first residue-resolution mechanistic study involving chromogranin peptides and their target proteins. Our approaches should be applicable to interaction studies involving other chromogranin-derived peptides and their cellular target proteins.