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Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultive aerobe Shewanella oniedensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 11.5 mg of cytochrome/g of cells.
Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 11.5 mg of cytochrome/g of cells.
Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopontin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post-transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level.
The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is complex. In order to document the changes in plant metabolism due to the symbiosis, we utilized laser ablation electrospray ionization mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have altered nodule ultrastructure. Results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid (JA), correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+) and ineffective (nifH mutant, fix-) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. This study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.
Increased matrix metalloproteinase 9 (MMP9) expression is involved in delayed wound healing in diabetic foot ulcers. We created skin wounds in normal SD rats and STZ-induced diabetic SD rats, then we found protein levels of activator protein-1 (AP1), a crucial transcription factor to increase MMP9 transcription, as well as MMP9 was up-regulated in epithelium of diabetic skin tissues. Then, we evaluated the mRNA and protein stability of AP1 subunits C-FOS/C-Jun in HaCaT cells after high glucose treatment. Results showed that high glucose could increase protein stability of C-FOS and C-Jun. Additionally, high glucose also activated extracellular signaling-related kinase 1/2 (ERK1/2). ERK1/2 inhibitor could rescue phosphorylation of C-FOS and C-Jun, increased protein stability of C-Jun, and increased MMP9 expressions. Thus, our study demonstrated that high glucose could activate ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic skin and HaCaT cells.
When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ 2 C 2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.
The viscoelasticity of concentrated protein suspensions associates closely with the mixing efficiency and cleaning frequency of facility during high-protein food development. This study investigated the effects of thermal-mechanical treatment on the viscoelasticity of milk protein isolate (MPI) suspensions and their underlying mechanisms to develop protein ingredient with low viscoelasticity. MPI suspensions (20%) were treated at 25, 50 and 85°C for 10-60 min under constant shear (100 s -1 ), followed by storage at 4°C. The viscosity (η) of MPI suspension treated at 50°C and 85°C was similar to 1-10% as those treated at 25 ° C. After four days of storage at 4°C, η showed the least value in 50°C-treated samples compared to those at 25 ° C and 85 ° C. The η and storage modulus (G') was decreased with prolonged treatment at 25 and 50°C, whereas opposite trend was found in 85°C treated samples. Differential scanning calorimetry found proteins in 50°C treated samples had smaller enthalpy than those in the control and 25°C treated samples. Protein surface hydrophobicity was increased slightly from 25°C to 50°C, but remarkably in 85°C treated samples. Ultra-small angle x-ray scattering showed the radius of gyration (R-g) of casein micelle was similar to 38 nm at 25°C and 50°C treated samples but increased to similar to 44 nm 85°C treated samples with reduced compactness. A new sphere-like structure with R-g of 18 nm was generated in 85°C treated samples. These findings suggested modulating temperature during thermal-mechanical treatment is essential to alter protein structures and morphology for desirable rheological properties.
Natural photosystems couple light harvesting to charge separation using a ‘special pair’ of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C 2 -symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.
In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.
The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.
An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-C DNAJB1 ) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-C DNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-C DNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with La III , Ce III , and Nd III using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. We find that his result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG–XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.
Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are neurodegenerative disorders which affect mammals, including the human species, and arise after the conversion of the monomeric cellular prion protein (PrP{sup C}) into the aggregated scrapie form (PrP{sup Sc}). There is no therapy to treat TSEs and the identification of compounds that bind PrP{sup C}, preventing its conversion into PrP{sup Sc}, is a viable therapeutic strategy. We designed and synthesized six novel trimethoxy-benzamide compounds as anti-prion drug candidates. Molecular docking analyses predicted that all the derivatives bind to a hotspot region located in the PrP globular domain with very similar spatial orientation and interaction mode. Although none of the analogs inhibited in vitro-aggregation of recombinant PrP (rPrP) in a cell-free conversion assay, the RT-QuIC, compound 8a accelerated rPrP conversion into PrP{sup Sc}-like species. STD-NMR and ITC analyses indicated that both 8a and 8b bind to rPrP{sup 90–231}. These analogs were toxic to PrP{sup Sc}-infected cell lines, hence we could not assess their anti-prion activity by using this cellular approach, although this toxicity was cell line-dependent. These results point out that the 4-amino-quinoline trimethoxy-benzamide scaffold described herein represents a novel chemical pattern useful as a starting point for future structural optimization in the design of PrP ligands with improved affinity and safety profiles.
The computational design of a symmetric protein homo-oligomer that binds a symmetry-matched small molecule larger than a metal ion has not yet been achieved. We used de novo protein design to create a homo-trimeric protein that binds the C 3 symmetric small molecule drug amantadine with each protein monomer making identical interactions with each face of the small molecule. Solution NMR data show that the protein has regular three-fold symmetry and undergoes localized structural changes upon ligand binding. A high-resolution X-ray structure reveals a close overall match to the design model with the exception of water molecules in the amantadine binding site not included in the Rosetta design calculations, and a neutron structure provides experimental validation of the computationally designed hydrogen-bond networks. Exploration of approaches to generate a small molecule inducible homo-trimerization system based on the design highlight challenges that must be overcome to computationally design such systems.
A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.
Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.
The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.