Molecular expression of adiponectin in human saliva

Adiponectin (APN) is an adipocyte-specific secretory protein that is highly and specifically expressed in adipose tissue. Serum APN consists of trimers, hexamers, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be of more bioactive forms. Evidence indicates that APN is produced by salivary gland epithelial cells, might be implicated in the regulation of local immune responses.     

Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization

The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far.Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.     

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.     

Influence of tyrosine-kinase Wzc activity on colanic acid production in Escherichia coli K12 cells

Bacterial tyrosine-kinases have been demonstrated to participate in the regulation of capsule polysaccharides (CPS) and exopolysaccharides (EPS) production and export. However, discrepant data have been reported on the molecular mechanism responsible for this regulation depending on the bacterial species analyzed. Special attention was previously paid to the tyrosine-kinase Wzc_ca of Escherichia coli K-12, which is involved in the production of the exopolysaccharide, colanic acid, and autophosphorylates by using a cooperative two-step process. In this work, we took advantage of these observations to investigate in further detail the effect of Wzc_ca phosphorylation on the colanic acid production. First, it is shown that expression of the phosphorylated form of Wzc prevents production of colanic acid whereas expression of the non-phosphorylated form allows biosynthesis of this exopolysaccharide. However, we provide evidence that, in the latter case, the size distribution of the colanic acid polymer is less scattered than in the case of the wild-type strain expressing both phosphorylated and non-phosphorylated forms of Wzc. It is then demonstrated that colanic acid production is not merely regulated by an on/off mechanism and that, instead, both phosphorylated and non-phosphorylated forms of Wzc are required to promote colanic acid synthesis. Moreover, a series of data suggests that besides the involvement of phosphorylated and non-phosphorylated forms of Wzc in the production of colanic acid, two particular regions of this kinase play as such an important role in the synthesis of this exopolysaccharide: a proline-rich domain located in the N-terminal part of Wzc_ca, and a tyrosine cluster present in the C-terminal portion of the enzyme. Furthermore, considering that polysaccharides are known to facilitate bacterial resistance to certain environmental stresses, it is shown that the resistance of E. coli to desiccation is directly connected with the phosphorylation state of Wzc_ca.     

The oligomeric conformation of peroxiredoxins links redox state to function

Protein-protein associations, i.e. formation of permanent or transient protein complexes, are essential for protein functionality and regulation within the cellular context. Peroxiredoxins (Prx) undergo major redox-dependent conformational changes and the dynamics are linked to functional switches. While a large number of investigations have addressed the principles and functions of Prx oligomerization, understanding of the diverse in vivo roles of this conserved redox-dependent feature of Prx is slowly emerging. The review summarizes studies on Prx oligomerization, its tight connection to the redox state, and the knowledge and hypotheses on its physiological function in the cell as peroxidase, chaperone, binding partner, enzyme activator and/or redox sensor.     

Effect of dietary fenugreek seeds on biliary proteins that influence nucleation of cholesterol crystals in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallising and anticrystallising factors present in bile. Dietary fenugreek seed has been recently observed to possess anti-lithogenic potential in experimental mice. In the current animal study, we evaluated the effect of dietary fenugreek on the compositional changes in the bile, particularly its effect on glycoproteins, low-molecular-weight (LMW) and high-molecular-weight (HMW) proteins, cholesterol nucleation time and cholesterol crystal growth. Groups of Wistar rats were fed for 10 weeks with diets: (1) basal control (C), (2) C + fenugreek (12%), (3) high cholesterol diet (HCD) and (4) HCD + fenugreek (12%). Feeding of HCD containing 0.5% cholesterol for 10 weeks rendered the bile lithogenic. Incorporation of fenugreek into HCD decreased the cholesterol content (70.5%), total protein (58.3%), glycoprotein (27.5%), lipid peroxides (13.6%) and cholesterol saturation index (from 1.98 to 0.75) in bile, increased the bile flow rate (19.5%), prolonged the cholesterol nucleation time and reduced the vesicular form of cholesterol (65%), which was accompanied with an increase in smaller vesicular form (94%). There was an increase in biliary phospholipid (33%) and total bile acid (49%) contents in the HCD + fenugreek group as compared with the HCD group. Electrophoretic separation of biliary LMW proteins showed the presence of a high concentration of 28-kDa protein, which might be responsible for the prolongation of cholesterol nucleation time in the fenugreek-fed groups. These findings indicate that the beneficial anti-lithogenic effect of dietary fenugreek, which primarily is due to reduction in the cholesterol content in bile, was additionally affected through a modulation of the nucleating and anti-nucleating proteins, which, in turn, affect cholesterol crystallisation.     

Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.     

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.     

Expression and role of adiponectin receptor 1 in lipopolysaccharide‐induced proliferation of cultured rat adventitial fibroblasts

Adiponectin is an adipose‐derived hormone that has anti‐diabetic and anti‐atherogenic effects through interaction with AdipoR1 and AdipoR2 (adiponectin receptors 1 and 2), but little is known about the expression and function of adiponectin and its receptors in adventitia and adventitial fibroblasts. In the present study, we have demonstrated that AdipoR1 is highly expressed in rat adventitia and cultured adventitial fibroblasts by quantitative real‐time PCR, Western blotting and immunofluorescent staining, whereas Adipo2 is low‐expressed. The expression of AdipoR1 have been observed to decrease gradually in adventitial fibroblasts in response to LPS (lipopolysaccharide) treatment. No local expression of adiponectin has been detected in adventitial tissues, indicating that serum adiponectin is the ligand for AdipoR1 in adventitial fibroblasts. In addition, treatment of recombinant adiponectin inhibited LPS‐induced proliferation of adventitial fibroblasts via activation of the AMPK (adenosine monophosphate‐activated protein kinase). AdipoR1 siRNA (small interfering RNA) transfection potently knocked down the receptor protein. The siRNA‐AdipoR1 transfected cells and AMPK inhibitor compound C treated cells showed decreased phosphorylated level of AMPK as determined by Western blot analysis, and increased the proliferation of adventitial fibroblasts as determined by BrdU (5‐bromo‐2′‐deoxyuridine) staining. These results demonstrated that adiponectin stimulates the proliferation of adventitial fibroblasts via the AdipoR1 and AMPK signalling pathways.     

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin,by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.     

Cloning,expression, and characterization of miR058 and its target PPO during the development of grapevine berry stone

Polyphenol oxidases catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms. They are key enzymes playing a significant role during the synthesis of lignin. The inhibition of the synthesis of lignin in grapevine can cause seedless grapevine berry development. In this study, grapevine PPO (Vv-PPO) was predicted as the target gene of Vv-miR058 by bioinformatics analysis, and it was further cloned and its homologous conservation in various plants was analyzed. The expression profiles of miR058 and its target Vv-PPO were detected by qRT-PCR in peel, pulp and seeds of three grapevine cultivars and Vv-PPO was expressed in an opposite variation way with Vv-miR058 where both of them could be detected, suggesting that Vv-miR058 can play an important role by regulating the expression of Vv-PPO. In addition, the potential target gene Vv-PPO for Vv-miR058 was verified by RLM-RACE. This result would be helpful in theoretical basis for further research and seedless grapevine berry production.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

Isolation and characterization of Glu-1 genes from the St genome of Pseudoroegneria libanotica

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.     

Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins

Low molecular weight juvenile hormone binding proteins (JHBPs) are specific carriers of juvenile hormone (JH) in the hemolymph of butterflies and moths. As hormonal signal transmitters, these proteins exert a profound effect on insect development. The crystal structure of JHBP from Galleria mellonella shows an unusual fold consisting of a long α-helix wrapped in a highly curved antiparallel β-sheet. JHBP structurally resembles the folding pattern found in tandem repeats in some mammalian lipid-binding proteins, with similar organization of one cavity and a disulfide bond between the long helix and the β-sheet. JHBP reveals, therefore, an archetypal fold used by nature for hydrophobic ligand binding. The JHBP molecule possesses two hydrophobic cavities. Several lines of experimental evidence conclusively indicate that JHBP binds JH in only one cavity, close to the N- and C-termini, and that this binding induces a structural change. The second cavity, located at the opposite end of the molecule, could bind another ligand.     

Isolation and Properties of Alginate Lyase from the Mid-Gut Gland of Wreath Shell Turbo cornutus

Alginate lyase [EC 4.2.2.3] has been purified from mid-gut gland of wreath shell. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. A molecular weight of 32,000 was estimated by SDS-polyacrylamide gel electrophoresis. Absorption spectra of the reaction products indicated that the enzyme had lyase activity. The enzyme was most active at a pH range of about 8.8 to 9.2 and most stable at pH 5 to 6. Phosphate showed strong stabilizing and enhancing effects on the enzyme activity. Divalent cations behaved differently toward alginic acid and propylene glycol alginate, suggesting requirements for free carboxyl groups and a single glycosidic chain in the enzyme action.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.     

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation.     

Identification and characterization of follistatin as a novel angiogenin-binding protein

Angiogenin enhances tumorigenesis. However, the mechanisms of angiogenin-induced angiogenesis and cancer cell proliferation remain elusive. In this study, follistatin was identified as a binding partner of angiogenin by a yeast two-hybrid screen and confirmed by a pull-down experiment. The interaction of fluorescently tagged angiogenin and follistatin was monitored in real time by a laser confocal microscope and shown to localize at the sub-nuclear region of HeLa cells. Additional yeast two-hybrid analysis revealed that domains 2 and 3 of follistatin were the minimal structure requirement for angiogenin binding. These findings provide new clues for further studies on the mechanisms of angiogenin-induced angiogenesis or cancer cell growth.