The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.
Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.
The effects of glucose and tolbutamide on the sodium handling of the pancreatic beta-cells were evaluated by measuring the total sodium content in intact islets from ob/ob-mice by integrating flame photometry and the free ion in individual beta-cells by dual wavelength fluorometry. Whereas increasing the glucose concentration from 3 to 20 mM resulted in a lowering of sodium, the addition of 100 microM tolbutamide caused a rise. The above-mentioned effects were most marked (about 50%) for the physiologically significant free sodium. The data indicate a more important role for Na+ in the regulation of insulin release than so far acknowledged. Increase of Na+ may contribute to the secretory response to hypoglycemic sulfonylureas by providing an additional rise of cytoplasmic Ca2+.
Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.
Cellular receptor systems are expected to present complex ligand interaction patterns that cannot beevaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluatedheterogeneous interactions using an alternative method to regression analysis, called Interaction Map(IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic,scalar kinetic association rate constant ka and dissociation rate constant kd with a two-dimensionaldistribution of ka and kd, it is possible to display heterogeneous data as a map where each peakcorresponds to one of the components that contribute to the cumulative binding curve. Here we challengethe Interaction Map approach by artificially generating heterogeneous data from two known interactions,on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM toaccurately decompose these man-made heterogeneous binding curves composed of two different interactions.We conclude that the Interaction Map approach is well suited for the analysis of complex bindingdata and forecast that it has a potential to resolve previously uninterpretable data, in particular thosegenerated in cell-based assays.
Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel expressed in epithelial cells. The effects of genistein and 4-phenylbutyrate (PBA) on CFTR were studied in three human airway epithelial cell lines expressing wild-type or DeltaF508 CFTR: Calu-3, CFSMEo-, and CFBE41o- cells. The cells were loaded with the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and chloride efflux was studied. Forskolin and 3-isobutyl-1-methylxanthine (IBMX) induced chloride efflux in Calu-3 cells but not in CF lines. Genistein (2.5-50 microM) alone was able to induce chloride efflux in all cell lines. Genistein did not enhance the effect of forskolin and IBMX. PBA had little or no effect on genistein-induced chloride efflux. The effect of genistein seen at low concentrations makes genistein interesting for possible pharmacological treatment of CF, since it is known that similar concentrations can be obtained in plasma by a soy-rich diet.
The malignant childhood brain tumor medulloblastoma belongs to the group of primitive neuroectodermal tumours (PNETs). Medulloblastomas are thought to arise from remnants of the transient external germinal layer in the cerebellum. Proliferation, differentiation, and motility of cells in the central nervous system are regulated by growth factors, e.g., platelet-derived growth factor (PDGF). Recently, it was shown that higher level of PDGF α-receptor expression is characteristic of metastatic medulloblastomas. We have investigated five medulloblastoma/PNET cell lines and found that the PDGF α-receptor is actively signalling in most of them, an activity most likely driven by endogenously produced PDGF-C. PDGF-C is normally present in cells of the developing external germinal layer and our results are consistent with the idea that medulloblastomas are derived from such cells undergoing early neuronal differentiation. Moreover, the expression of PDGF and its receptors was associated with neuronal characteristics, but not with high levels of c-myc expression in the medullablastoma cells.
Free radicals have been shown to be involved in bone resorption in vitro and in rodents. We studied the effect of oxidative stress on bone mineral density (BMD) in 48 women and 53 men from a population-based study. The levels of 8-iso-PGF(2alpha) (a major F(2)-isoprostane and a biomarker of oxidative stress) and a control, 15-keto-dihydro-PGF(2alpha) (a biomarker of inflammatory response), were measured in urinary samples and their association with BMD and quantitative ultrasound (QUS) measurements were examined. In multivariate linear regression analyses, 8-iso-PGF(2alpha) levels were negatively associated with both BMD and QUS. In contrast, no association was found for 15-keto-dihydro-PGF(2alpha). Our findings establish a biochemical link between increased oxidative stress and reduced bone density and provide a rational for further studies investigating the role of pro- and antioxidants in osteoporosis. Copyright 2001 Academic Press.
We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.
Misregulation of the Wnt/beta-catenin signalling pathway is involved in the development and progression of many cancers. Recently, we presented evidence for aberrant accumulation of non-phosphorylated (stabilized) beta-catenin in benign parathyroid tumors from patients with primary hyperparathyroidism (pHPT) or HPT secondary to uremia (sHPT). Here we have used a human parathyroid hormone (PTH)-producing cell line (sHPT-1), established from a hyperplastic parathyroid gland removed at operation of a patient with sHPT, to further investigate the potential importance of beta-catenin in parathyroid tumorigenesis. Our studies demonstrate that efficient and specific knockdown of beta-catenin by small interfering RNA (siRNA) markedly decreased endogenous beta-catenin transcriptional activity as well as expression of the Wnt/beta-catenin target genes cyclin D1 and c-myc, known to be overexpressed in a substantial fraction of parathyroid tumors. Furthermore, siRNA to beta-catenin inhibited cellular growth and induced cell death. Growth and survival of the parathyroid tumor cells are thus dependent on maintained expression level of beta-catenin. The Wnt/beta-catenin signalling pathway, and beta-catenin in particular, presents a potential therapeutic target for HPT.
Autoimmune polyendocrine syndrome type I (APS I) is a rare hereditary condition considered a model disease for organ specific autoimmunity. A wide range of autoantibodies targeting antigens present in the affected organs have been identified. Autoantibodies against aromatic l-amino acid decarboxylase (AADC) are present in about 50% of APS I patients. In order to increase our understanding of autoantibody specificity in APS I, the aim of the present study was to localize target regions on AADC recognized by sera from APS I patients. Using several complementing strategies, we have shown that autoantibodies against AADC mainly recognize conformational epitopes. The major antigenic determinants were detected N-terminally to amino acid residue 237. Replacement of amino acids 227–230 (ERDK) with alanine residues reduced the reactivity towards AADC by >80% in all patient sera tested, suggesting that amino acids 227–230 are an important part of an immunodominant epitope.
Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and has previously been shown to regulate bone mass by inhibiting osteoclast differentiation and activation. Recent evidence indicates that OPG also plays a role in the vascular system, since ablation of the OPG gene in mice results in calcification of the aorta and renal arteries, and association has been found between serum levels of OPG and cardiovascular mortality. This study presents a novel single nucleotide polymorphism, a T/C transition located 129 bp upstream the TATA-box of the human OPG gene, detected by sequence analysis. The OPG genotype was determined by restriction fragment length polymorphism in a cohort consisting of 59 healthy subjects. The intima-media thickness (IMT) in the common carotid artery and maximal post-ischemic forearm blood flow (FBF) were investigated. Subjects with the CC genotype showed a significantly increased IMT (p<0.05) and a concommitantly reduced maximal FBF (p<0.01) as compared to those with the T allele. Thus, our results show that the polymorphism in the promoter region of OPG is associated with both vascular morphology and function in apparently healthy subjects.
Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.
The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to β-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of β-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to β-sheet structure that coincides with the formation of α-synuclein oligomers. Albeit through different kinetic pathways depending on the degree of cross-linking.
Obestatin is a peptide hormone that is derived from the same polypeptide precursor (preprogrelin) as ghrelin, but it acts in opposing way on ingestive behavior. Our previous studies showed that ghrelin affects memory and anxiety. Here, we studied the possible effects of icv obestatin injection in rats upon memory retention (using two different paradigms), anxiety like behavior (plus maze test), and food intake. Obestatin induces an increase in the percentage of open arms entries (Obestatin 3.0 nmol/rat: 61.74 ± 1.81), and percentage of time spent in open arms (Obestatin 3.0 nmol/rat: 72.07 ± 4.21) in relation to the control (33.31 ± 1.54; 25.82 ± 1.68), indicating an anxiolytic effect. The two doses of obestatin increased latency time in a step down test and the percentage time of novel object exploration, suggesting that the peptide influences learning and memory processes that involve the hippocampus and the amygdala. This report provides evidence indicating that obestatin effects are functionally opposite on anxiety and hunger to the ghrelin effects, while both these related peptides increase memory retention.
Solute carriers (SLCs), the second largest super-family of membrane proteins in the human genome, transport amino acids, sugars, fatty acids, inorganic ions, essential metals and drugs over membranes. To date no study has provided a comprehensive analysis of SLC localization along the entire GI tract. The aim of the present study was to provide a comprehensive, segment-specific description of the localization of SLC genes along the rat Cl tract by employing bioinformatics and molecular biology methods. The Unigene database was screened for rat SLC entries in the intestinal tissue. Using qPCR we measured expression of the annotated genes in the Cl tract divided into the following segments: the esophagus, the corpus and the antrum of the stomach, the proximal and distal parts of the duodenum, ileum, jejunum and colon, and the cecum. Our Unigene-derived gene pool was expanded with data from in-house tissue panels and a literature search. We found 44 out of 78 (56%) of gut SLC transcripts to be expressed in all Cl tract segments, whereas the majority of remaining SLCs were detected in more than five segments. SLCs are predominantly expressed in gut regions with absorptive functions although expression was also found in segments unrelated to absorption. The proximal jejunum had the highest number of differentially expressed SLCs. In conclusion, SLCs are a crucial molecular component of the Cl tract, with many of them expressed along the entire GI tract. This work presents the first overall road map of localization of transporter genes in the Cl tract.
Towards repairing bone defects, calcium sulfate and calcium phosphate cement have been recognized as promising bone grafts. However, the current bone cements are generally lack of proper porosity for cell migration and new tissue formation. On the other hand, porous scaffold cannot be delivered by injection, which limits its use its clinical use. Herein, we develop a novel tricalcium phosphate/calcium sulfate granule to overcome the limitations of injectable cements and traditional scaffolds. The biocompatible granule underwent in situ self-setting to form scaffold with porous structure after injection. It con-tributes to calcium deposition and upregulation of osteogenic genes of mesenchymal stem cells in a time-dependent manner. Within three months, cavitary bone defects of distal rabbit femurs implanted the granules exhibited better bone formation than those with those implanted with autologous bone.
The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jimenez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.
Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their beta-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR beta-subunit (GlyR beta) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyR beta in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.
The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the beta-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7-higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.
Apolipoprotein A1 (apoA1) is the main protein component responsible for transportation of cholesterol on high-density lipoprotein (HDL). Serum amyloid A (SAA) is an acute phase protein associated with HDL. Apart from their physiological functions, both apoA1 and SAA have been identified as 'amyloidogenic peptides'. We report herein that the polysaccharide heparin interacts with both apoA1 and SAA in HDL isolated from plasma of inflamed mice. The reaction is rapid, forming complex aggregates composed of heparin, apoA1 and SAA as revealed by gel electrophoresis. This interaction is dependent on the size and concentration of added heparin. Mass spectrometry analysis of peptides derived from chemically crosslinked HDL-SAA particles detected multiple crosslinks between apoA1 and SAA, indicating close proximity (within 25 angstrom) of these two proteins on the HDL surface, providing a molecular and structural mechanism for the simultaneous binding of heparin to apoA1 and SAA.
Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLTI and CYSLT2. However, montelukast, an inhibitor of CYSLTI, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLTI signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. (C) 2017 The Author(s). Published by Elsevier Inc.
Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1beta, TNF-alpha, and IFN-gamma), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and beta cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.
One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.
HER2-specific affibody molecules in different formats have previously been shown to be useful tumor targeting agents for radionuclide-based imaging and therapy applications, but their biological effect on tumor cells is not well known. In this study, two dimeric ((ZHER2:4)2 and (ZHER2:342)2) and one monomeric (ZHER2:342) HER2-specific affibody molecules are investigated with respect to biological activity. Both (ZHER2:4)2 and (ZHER2:342)2 were found to decrease the growth rate of SKBR-3 cells to the same extent as the antibody trastuzumab. When the substances were removed, the cells treated with the dimeric affibody molecules continued to be growth suppressed while the cells treated with trastuzumab immediately resumed normal proliferation. The effects of ZHER2:342 were minor on both proliferation and cell signaling. The dimeric (ZHER2:4)2 and (ZHER2:342)2 both reduced growth of SKBR-3 cells and may prove therapeutically useful either by themselves or as carriers of radionuclides or other cytotoxic agents.
OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate → inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate → inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.
Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.
Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer's disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [3H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [3H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.
Previous studies have suggested that hepatic production of 25-hydroxyvitamin D3 may be suppressed by 1α,25-dihydroxyvitamin D3. However, the molecular details of these observations have not been clarified. In the current study, the 5´-flanking DNA sequence of CYP2D25, a porcine microsomal vitamin D 25-hydroxylase, was isolated and analyzed. The CYP2D25 promoter contains a putative vitamin D response element (VDRE). The promoter activity was markedly suppressed by 1α,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in presence of vitamin D receptor (VDR). The data suggest that VDR-mediated inhibition of 25-hydroxylase(s) by vitamin D3 metabolites at the transcriptional level may play an important role in the regulation of 25-hydroxyvitamin D3 production in liver and other tissues.
The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.
We recently described the atypical Rho GTPases Miro-1 and Miro-2. These proteins have tandem GTP-binding domains separated by a linker region with putative calcium-binding motives. In addition, the Miro GTPases have a C-terminal transmembrane domain, which confers targeting to the mitochondria. It was reported previously that a constitutively active mutant of Miro-1 induced a clustering of the mitochondria. This response can be separated into two distinct phenotypes: a formation of aggregated mitochondria and the appearance of thread-like mitochondria probably caused by defects in mitochondrial trafficking. The first GTPase domain is required for the clustering of the mitochondria, but the effect is not dependent on the EF-hands. Miro-2 only induces aggregation and not the formation of thread-like mitochondria. Moreover, we show that Miro interacts with the Kinesin-binding proteins, GRIF-1 and OIP106, suggesting that the Miro GTPases form a link between the mitochondria and the trafficking apparatus of the microtubules.
The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (FTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic beta-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of FIB to the 5'-UTR of the human insulin mRNA.
The aim of this study was to further elucidate the role of the polypyrimidine tract binding protein (PTB) in the control of insulin mRNA stability. We observed that the glucose- or interleukin-1β-induced increase in insulin mRNA was paralleled by an increase in PTB mRNA. To further test the hypothesis that PTB controls insulin gene expression, βTC-6 cells were treated with a PTB-specific siRNA to modify the β-cell content of PTB. Surprisingly, we observed an increase in PTB mRNA and PTB protein levels in response to the siRNA treatment. In addition, the PTB-siRNA treatment also increased insulin mRNA. We conclude that expression of the PTB gene controls insulin production.
We report six novel members of the superfamily of human G-protein coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR123, GPR124, GPR125, GPR126, GPR127, and GPR128. Phylogenetic analysis demonstrates that these are additional members of the family of GPCRs with long N-termini, previously termed EGF-7TM, LNB-7TM, B2 or LN-7TM, showing that there exist at least 30 such GPCRs in the human genome. Three of these receptors form their own phylogenetic cluster, while two other places in a cluster with the previously reported HE6 and GPR56 (TM7XN1) and one with EMR1-3. All the novel receptors have a GPS domain in their N-terminus, except GPR123, as well as long Ser/Thr rich regions forming mucin-like stalks. GPR124 and GPR125 have a leucine rich repeat (LRR), an immunoglobulin (Ig) domain, and a hormone-binding domain (HBD). The Ig domain shows similarities to motilin and titin, while the LRR domain shows similarities to LRIG1 and SLIT1-2. GPR127 has one EGF domain while GPR126 and GPR128 do not contain domains that are readily recognized in other proteins beyond the GPS domain. We found several human EST sequences for most of the receptors showing differential expression patterns, which may indicate that some of these receptors participate in central functions while others are more likely to have a role in the immune or reproductive systems.
Linoleate diol synthases (LDS) are homologous 8(R)-dioxygenases with hydroperoxide isomerase activities, expressed in fungal pathogens of humanitarian importance. We report for the first time expression and site-directed mutagenesis of LDS. 7,8-LDS of the take-all fungus, expressed in Pichia pastoris, oxygenated 18:2n − 6 to 8(R)-hydroperoxylinoleic acid, which was unexpectedly isomerized to 5,8(R)-dihydroxylinoleic acid (60% 5S) and to 8(R),13-dihydroxyoctadeca-9(E),11(E)-dienoic acid. The latter was likely formed via hydrolysis of an unstable intermediate, 8(R),9(S)-epoxyoctadeca-10(E),12(Z)-dienoic acid. A tyrosyl radical is formed during 7,8-LDS catalysis, and Tyr376 is the sequence homolog to Tyr385 of cyclooxygenase-1. Tyr376Phe retained hydroperoxide isomerase activity but lacked 8(R)-dioxygenase activity. The putative proximal heme ligand His379 and the N-glycosylation site at Asn216 appeared to be critical for 8(R)-dioxygenase activity, as His379Gln and Asn216Gln were inactive. Treatment with α-mannosidase to shorten N- and O-linked mannosides inhibited the hydroperoxide isomerase but not the 8(R)-dioxygenase. Our results suggest that post-translational modifications may influence the oxidation mechanism of 7,8-LDS.
Retinoids have been shown to modulate inflammation and the immune response in many cell types including macrophages, endothelial cells, and vascular smooth muscle cells. However, present knowledge of whether inflammatory mediators modulate vitamin A status in these cells is limited. To identify the role of inflammation on retinoid metabolism in vascular smooth muscle cells, the cells were exposed to a combination of proinflammatory cytokines: interleukin-1beta, interferon-gamma, and lipopolysaccharides. Without stimulation with proinflammatory cytokines, vascular smooth muscle cells expressed retinol dehydrogenases-2 and 5 mRNA detected by RT-PCR. Stimulation with the combination of cytokines induced a substantial increase of retinol dehydrogenase-5 mRNA. This was associated with increased production of ligands for retinoic acid receptors, when assayed in a retinoic acid receptor-dependent luciferase reporter system. Our results demonstrate that inflammatory mediators activate the retinoid metabolic pathway in vascular smooth muscle cells, which potentially may modulate the inflammatory response in the vascular wall.
Anterior Gradient 2 (AGR2) is a potential anti-tumor target and we previously reported a murine antibody 18A4 with specific binding to AGR2. However, humanization is a must to overcome immunogenicity before considering for clinical use and optimized vectors for mammalian expression are also necessary for following industrialized manufacture. Here, we describe an anti-tumor humanized antibody blocking secreted AGR2 activity. We employed the CDR grafting technique and deimmunization analysis to construct humanized antibody variants of 18A4, and 18A4Hu I was selected as the best humanization candidate, characterized by physical and chemical property comparison. Mouse xenograft study showed that 18A4Hu I could effectively inhibit the xenograft tumor growth, antibody blocking epitope analysis using AGR2 mutants indicated that the inhibition activity of 18A4Hu I is exerted probably through blocking the AGR2 functions which rely on the amino acid sites of E60-H76 and A86-E153. What's more, in this report, we also describe a pHAb-FAST vector system which is specifically designed for humanized antibody mammalian expression vector fast construction. With pHAb-FAST system, expression vector of 18A4Hu I could be quickly constructed only through twice overlapping PCR reactions. To our knowledge, AGR2-targeted 18A4Hu I is a promising humanized anti-tumor drug candidate, and pHAb-FAST system is a useful optimized mammalian expression vector construction tool. Our findings are supposed to accelerate the development of antibody-based cancer therapy.
Using a quantitative single nucleotide polymorphism (SNP) assay we have investigated the changes in the expression of the BCR-ABL1 oncogene relative to the wild-type ABL1 and BCR alleles in cells from chronic myeloid leukemia (CML) patients not responding to therapy. The results show a progressive increase in the BCR-ABL1 oncogene expression at the expense of decreased expression of the ABL1 allele, not involved in the fusion. No relative changes in the expression of the two BCR alleles were found. These results demonstrate that allele-specific charities in gene expression, with selective, progressive silencing of the wild-type A BL1 allele in favor of the oncogenic BCR-ABL1 allele occur in CML patients with therapy-resistant disease.
Semicarbazide-sensitive amine oxidase (SSAO) activity in plasma is increased in diabetes, and in particular, in diabetic patients with vascular complications. It has been speculated that SSAO is involved in the development of such complications due to the production of cytotoxic compounds. In this work, we have induced diabetes in a previously described mouse-model, overexpressing SSAO in smooth muscle cells. SSAO activity was estimated as well as expression of the endogenous mouse gene and human transgene using real-time PCR. Diabetes induced an increase in SSAO activity in serum, kidney, and adipose tissue of transgenic animals. An inverse correlation between SSAO activity and mouse SSAO mRNA levels was observed in transgenic animals with diabetes. These results further support the suggestion of a negative feedback control of the SSAO gene expression. The increased SSAO activity in diabetes is most likely dependent on post-transcriptional modifications or activation of existing inactive enzyme molecules.
Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P-3 levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P-3 and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.
Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca2+([Ca2+](i)) below baseline in pancreatic beta-cells. The period of [Ca2+], decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic alpha(2A) and muscarinic M-3 receptors affects the initial [Ca2+](i) response to increase of glucose from 3 to 20 mM in beta-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca2+](i) during 90-120 s followed by rise due to opening of voltage-dependent Ca2+ channels. The period of [Ca2+](i) decrease was prolonged by activation of the alpha 2A adrenergic receptors (1 mu M epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M-3 receptors (0.1 mu.M acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10-100 mu M). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca2+](i) rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca2+](i); regulates the subsequent beta-cell response to glucose.(C) 2014 Elsevier Inc. All rights reserved.
Recent studies of isolated human islets have shown that glucose induces hormone release with repetitive pulses of insulin and somatostatin in antisynchrony with those of glucagon. Since the mouse is the most important animal model we studied the temporal relation between hormones released from mouse islets. Batches of 5-10 islets were perifused and the hormones measured with radioimmunoassay in 30 s fractions. At 3 mM glucose, hormone secretion was stable with no detectable pulses of glucagon, insulin or somatostatin. Increase of glucose to 20 mM resulted in an early secretory phase with a glucagon peak followed by peaks of insulin and somatostatin. Subsequent hormone secretion was pulsatile with a periodicity of 5 min. Cross-correlation analyses showed that the glucagon pulses were antisynchronous to those of insulin and somatostatin. In contrast to the marked stimulation of insulin and somatostatin secretion, the pulsatility resulted in inhibition of overall glucagon release. The cytoarchitecture of mouse islets differs from that of human islets, which may affect the interactions between the hormone-producing cells. Although indicating that paracrine regulation is important for the characteristic patterns of pulsatile hormone secretion, the mouse data mimic those of human islets with more than 20-fold variations of the insulin/glucagon ratio. The data indicate that the mouse serves as an appropriate animal model for studying the temporal relation between the islet hormones controlling glucose production in the liver.
Studies of insulin producing β-cells have reported conflicting responses to NF-κB activation,encompassing both pro- and anti-apoptotic effects, possibly reflecting the use of β-cells fromdifferent species. Therefore, the aim of this study was to compare the temporal activation of NF-κB in rat and human insulin producing cells and relate this to the dynamics of cell death, STAT-1activation and the production of nitric oxide (NO). Rat RIN5AH and human islet cells wereexposed to the cytokines IL-1β and IFN-γ and the NOS inhibitor aminoguanidine. Cell death, NOproduction, ΙκΒα phosphorylation, p65 methylation, STAT-1 phosphorylation and cIAP-2 levelswere analyzed at different time-points. Cytokine-induced RIN5AH cell death occurred on day 1,and this was paralleled by NF-κB activation, STAT-1 phosphorylation and production of NO. Onthe other hand, the human islet cells instead died by an NO-independent mechanism on day 3and 5. This later occurring cell death was associated with a gradual decrease in ΙκΒα phosphorylation and p65 methylation, and a lowered expression of the NF-κB target genes ΙκΒα and cIAP-2. STAT-1 phosphorylation was persistently high during the entire cytokine exposureperiod in human islet cells. The results favor a pro-survival role of NF-κB and a pro-apoptoticrole of STAT-1 in human islet cells. Thus, rodent insulin producing cells may not be suitable asmodels for human β-cells in the context of cytokine-induced damage.
Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D3 due to decreased 25-hydroxylation of vitamin D3 in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D3 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.
There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).
Although the hepatitis C virus (HCV) genome is synthesized by the virus-encoded RNA-dependent RNA polymerase NS5B, other viral and cellular factors are assumed to be required for template-specific initiation and regulation of RNA-synthesis. The cellular protein La, which normally associates with RNA polymerase III transcripts, also interacts with the 5'- and 3'-untranslated regions of several RNA viruses, including HCV. To investigate whether other viral gene products may be involved in this interaction, we constructed an HCV cDNA expression library in bacteriophage T7 allowing portions of the HCV polyprotein to be displayed on the phage surface. Screening of the phage library against La resulted in selection of clones displaying the N-terminal region of HCV NS5A. Co-precipitation of full-length and truncated forms of recombinant NS5A with La revealed that the N-terminal region of NS5A was both necessary and sufficient for binding to La. Although this region of NS5A is essential for HCV replication, the role of the NS5A-La interaction in the infected cell remains to be established.
Transgenic mice overexpressing fibroblast growth factor 23 (FGF23) in osteoblasts have a rachitic bone phenotype. These mice display hypomineralized bones, increased expression of osteoblast markers, but osteoclast numbers are unaltered or slightly reduced. Paradoxically, they show increased serum levels of the bone resorption marker CTX, a type I collagen degradation fragment. Here we analyzed a matrix metalloproteinase- (MMP-) like secreted protease, Adamts1, that has previously been associated with osteoblastic type I collagen breakdown in vitro. Bones from FGF23 transgenic (tg) mice displayed increased Adamts1 protein upon both immunohistological staining and Western blotting. We further found Adamts1 protein together with excessively degraded type I collagen in the non-mineralized bone fraction of FGF23 tg mice. A similar degradation pattern of type I collagen was noticed upon forced expression of Adamts1 in osteoblastic cells in vitro. Importantly, these Adamts1-expressing osteoblastic cells exhibited increased release of CTX fragments when cultured on demineralized bone discs. Together, these results demonstrate for the first time that Adamts1 can be highly induced in bone tissue and that this MMP-like protease can increase osteoblastic release of CTX fragments from non-mineralized bone. Thus, Adamts1 potentially contributes to the increased serum levels of CTX in rickets/osteomalacia.
Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.
Rat liver fructose-1,6-diphosphatase was phosphorylated with (32P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(32P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent Km of 400 μM, while the apparent Km value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The Vmax value was 20 times higher for the peptide than for the enzyme.