Critical Role of Cys168 in Noggin Protein's Biological Function

Previous studies have indicated that noggin exerts its neural inducing effect by binding andantagonizing bone morphogenetic protein 4(BMP4).In order to further clarify the relationship between thestructure and the function of noggin,and elucidate the possible mechanism responsible for noggin-BMP4interaction,we generated three noggin mutants,C168S,C174S and C197S,by using a site-directed mu-tagenesis method.Ectopic expression of wild-type(WT)noggin,C174S or C197S,in Xenopus animal caps(ACs)by mRNA injection converted the explants(prospective ectoderm)into neural tissue,as indicated bythe neural-like morphology and expression of the neural cell adhesion molecule(NCAM)in the ACs.Incontrast,ACs expressing C 168S suffered an epidermal fate similar to the control caps.Similarly,among the threemutants,only C 168S lost the dorsalizing function.These studies highlight the critical role played by Cys168in noggin's biological activities.It probably participates in the formation of an intermolecular disulfide bridge.     

Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

AIM: To identify alkyl hydroperoxide reductase subunit C(AhpC) homologs in Bacillus subtilis(B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.METHODS: Two AhpC homologs(AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues(C37S, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.     

Functional analysis of three amino acid residues of purR repressor, Trp147, Gln-218 and Gln-292 in Salmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Identification of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88 genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes were firstly associated with UL. Three genes with notable difference were selected for Northern confirmation. Our results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate, testis, liver, heart and skeletal muscle.     

Identification of up—regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas(Uls),suppression sub-tractive hybridization was used to construct an UL up-regulated library,which turned out to represent 88 genes.After two rounds of screening by reverse Northern analysis,twenty genes were proved to be up-regulated,including seventeen known genes and three genes with unknown function.All these genes were firstly associated with UL.Three genes with notable difference were selected for Northern confirmation.Our results proved the authenticity of the twenty genes.One gene named Phospholipase A2(PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate,testis,liver,heart and skeletal muscle.     

Characterization of the functionally related sites in the neural inducing gene noggin

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.     

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.     

Impact of cysteine variants on the structure,activity, and stability of recombinant human α-galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.     

The BMP antagonists cerberus-like and noggin do not interact during mouse forebrain development

Mouse cerberus-like encodes for a secreted factor of the Cerberus/Dan family. This molecule has neural inducing capabilities and can bind to BMP-4 and nodal molecules in the extracellular space. When cerberus-like is inactivated, its function may be compensated for another molecule, since no abnormalities can be observed in the mouse mutant. Compensation mechanisms have been shown to occur between the BMP antagonists chordin and noggin. Here we report the generation of cerberus-like-/-; noggin-/- double mutants to uncover a possible compensation by noggin in cer-l-/- mutant. Double mutants were obtained and failed to show any further detectable defects beside the ones presented by the noggin-/- single mutant. Contrarily to chordin and noggin, mouse cerberus-like and noggin cannot compensate for each other during mouse embryogenesis.     

Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP

Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP.