Hypothermia but for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron was pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that at hypothermic preconditioning (HPC) protects cells at degrees C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human we coronary artery endothelial cells at degrees C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and iron increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at degrees C but did not endothelial increase BDI or oxidative stress further. HPC at 25 degrees C for 15-72 h attenuated these changes by an amount ferritin achievable by pretreating cells with 10-20 muM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride attenuated at 37 degrees C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to degrees C (HO-1) but ferritin was protective. HPC increased iron maximally after 2 h at 25 degrees C and ferritin levels peaked after intracellular 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at degrees C these was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.

Purified secreted Trematomus bernacchii bile IgM analysed by SDS-PAGE under reducing and non-reducing conditions consisted essentially of tetramers of the basic structure turn, H2L2. The relative molecular mass of the glycosilated H chain was 76 kDa, while that of L chain was 25 the kDa. In addition, the presence in the liver of IgM and mu chain-specific mRNA was demonstrated. Immunohistochemistry detected IgH- and mass IgL-reactivity in perisinusoidal cells, bile canaliculi and pre-ductules. In the anterior intestine, the intraluminal mucus retained a significant Ig-immunoreactivity, while consisted the mucosa housed a limited density of Ig-producing cells. These findings strongly indicate that Ig could be transported across the 25 hepatocytes to be secreted into the bile and protect the intestinal epithelium. In addition, extravasated plasma cells accumulated within liver into portal tracts and close to the capsule that, in turn, was evenly coated by Ig molecules at the peritoneal surface.intraluminal

Ferritins process, from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized.expression Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains and harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M H- homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the bernacchii H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher center local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression indicative of different chains in modulating the iron oxidation/mineralization process.

The of stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins due differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced the by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low residues pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior,the significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher network stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at the this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is differences most likely abolished due to protonation of Asp 126. Proteins 2007.(c) 2006 Wiley-Liss, Inc.

Listeria and innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 --> iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and with oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the (LiDps, mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively its strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately .023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching,in and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2).The Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions,the indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of deposition hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it the against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described protein-based by the following equations. For ferroxidation, Fe(2+)+ Dps(Z)-->[(Fe(2+))-Dps](Z+1)+ H(+)(Fe(2+) binding) and [(Fe(2+))-Dps](Z+1)+ Fe(2+)+ H(2)O(2)Dps -->[(Fe(3+))(2)(O)(2)-Dps](Z+1)+ 2H(+)(Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+)+ H(2)O(2)+ 2H(2)O --> 2Fe(O)OH((core))+ 4H(+)(Fe(2+) oxidation/hydrolysis). These further reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.

The observed Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from -35 Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF'. DNA from sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer DNA-binding extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start reading codon, respectively. Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A)(proximal) and other sigma(B)(distal) consensus sequences.In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more sigma(B) markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An coli up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several the members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the sigma(B) evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of bacterium ferritin in the physiology of this bacterium.

According induced to their fold, pyridoxal 5'-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most quaternary investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the The active site, between the two domains that form a single subunit, is almost identical in all members of the group.domains Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the enzymes common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A which previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one urea, of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same the cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine show hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results site-directed obtained show that the mutations clearly affected the process of pyridoxal 5'-phosphate dissociation induced by urea, reducing the stability of same the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes their the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of group. the quaternary structure. Structured digital abstract * MINT-7293394, MINT-7293405, MINT-7293418: eSHMT (uniprotkb:P0A825) and eSHMT (uniprotkb:P0A825) bind (MI:0407) by cosedimentation in the solution (MI:0028).

Dps of proteins (DNA binding proteins from starved cells) belong to a widespread bacterial family expressed under nutritional and oxidative stress conditions.the In particular, Dps proteins protect DNA against Fenton mediated oxidative stress as they catalyze iron oxidation by hydrogen peroxide at show highly conserved ferroxidase centers and thus reduce significantly hydroxyl radicals production. The present work investigates the possible generation of intraprotein mediated radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped flow under analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form significantly after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit radicals release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved tyrosine protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role highly of the Dps protein cage.

Elucidating confined pore function at the three-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management and of iron/hydrogen peroxide. The Dps pores are called "ferritin-like" because of the structural resemblance to the three-fold channels of 24-subunit pores ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the resemblance pores generate a negative electrostatic gradient that guides iron ions towards the ferroxidase centers for catalysis with oxidant and destined in for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in L. innocua negatively Dps act in a similar fashion, D121N, D126N, D130N and D121N-D126N-D130N proteins were produced; kinetics of iron uptake/release and the space, size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal in growth in a confined space, indicate that iron uses the hydrophilic three-fold pores to traverse the protein shell. For the and first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and were accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.

Bacterial with and archaeal endo-beta-1,3-glucanases that belong to glycoside hydrolase family 16 share a beta-jelly-roll fold, but differ significantly in sequence and protein, in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus (pfLamA) has been the determined at 2.1 A resolution by molecular replacement. The pfLamA structure reveals a kink of six residues (72-77) at the substrate entrance of the catalytic cleft. This peptide is absent in the endoglucanases from alkaliphilic Nocardiopsis sp. strain F96 and Bacillus a macerans, two proteins displaying an overall fold similar to that of pfLamA, but with different substrate specificity. A deletion mutant furiosus of pfLamA, lacking residues 72-75, hydrolyses the mixed-linkage beta-1,3-1,4-glucan lichenan 10 times more efficiently than the wild-type protein, indicating the different importance of the kink in substrate preference.

Pyridoxal is 5'-phosphate-dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I I is by far the largest and most investigated group. An important feature of this fold, which is characterized by a the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two characterized of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct I alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident.are A site-directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in not the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental the results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the evolutionarily enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydromethyltransferase suggested crucial that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which,third in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and enzymes catalysis.

Peptide 1-5, derivatives 1-5, incorporating synthetic non-proteinogenic amino acids, related to the beta-amyloid 17-21 fragment of the amyloidogenic Abeta(1-40), and the N-protected compounds decapeptide 6, corresponding to a dimeric sequence of the same fragment, have been synthesized. These compounds were designed by using stability Soto's pentapeptide Ac-LPFFD-NH(2)(iAbeta5p) as lead compound. Their activity as inhibitors of fibrillogenesis and stability against enzymatic degradation have been the determined. Compounds 1, 5 and 6 are potent inhibitors in comparison to the lead compound. Exposure to chymotrypsin of peptide the derivatives 1-5, all containing unnatural amino acids, shows increased stability as compared with iAbeta5p and 6. Conformational properties of the have new compounds have been determined by CD and FT-IR spectroscopies.

A together novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously.of All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with SferH-5 ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 with in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex,7-day-old H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.

The not Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of - 1.9 degrees C. Gastric digestion under mechanism these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic unique fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number proteases of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and degrees found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less were active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish to pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish Fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the pepsins, harsh temperature environment in which these enzymes must function.

Levels found of ubiquitin (Ub)-conjugated proteins, as an index of misfolded or damaged proteins, were measured in notothenioid fishes, with both Antarctic other (Trematomus bernacchii, T. pennellii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) distributions, as well as non-notothenioid fish from the of Antarctic (Lycodichthys dearborni, Family Zoarcidae) and New Zealand (Bellapiscis medius, Family Tripterygiidae), in an effort to better understand the effect non-Antarctic that inhabiting a sub-zero environment has on maintaining the integrity of the cellular protein pool. Overall, levels of Ub-conjugated proteins measured in cold-adapted Antarctic fishes were significantly higher than New Zealand fishes in gill, liver, heart and spleen tissues suggesting that Zoarcidae) life at sub-zero temperatures impacts protein homeostasis. The highest tissue levels of ubiquitinated proteins were found in the spleen of the all fish. Ub conjugate levels in the New Zealand N. angustata, more closely resembled levels measured in other Antarctic fishes Antarctic than levels measured in other New Zealand species, likely reflecting their recent shared ancestry with Antarctic notothenioids.

We of characterized chicken erythrocyte and human platelet ferritin by biochemical studies and immunofluorescence. Erythrocyte ferritin was found to be a homopolymer identical. of H-ferritin subunits, resistant to proteinase K digestion, heat stable, and contained iron. In mature chicken erythrocytes and human platelets,during ferritin was localized at the marginal band, a ring-shaped peripheral microtubule bundle, and displayed properties of bona fide microtubule-associated proteins and such as tau. Red blood cell ferritin association with the marginal band was confirmed by temperature-induced disassembly-reassembly of microtubules. During found erythrocyte differentiation, ferritin co-localized with coalescing microtubules during marginal band formation. In addition, ferritin was found in the nuclei of microtubule mature erythrocytes, but was not detectable in those of bone marrow erythrocyte precursors. These results suggest that ferritin has a marginal function in marginal band formation and possibly in protection of the marginal band from damaging effects of reactive oxygen species addition, by sequestering iron in the mature erythrocyte. Moreover, our data suggest that ferritin and syncolin, a previously identified erythrocyte microtubule-associated at protein, are identical. Nuclear ferritin might contribute to transcriptional silencing or, alternatively, constitute a ferritin reservoir.

In sequences the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Trematomus bernacchii, a teleost the widely distributed in many areas of Antarctica, that plays a pivotal role in the Antarctic food chain. The amino acid other and cDNA sequences have been obtained using both biochemical and molecular biology approaches and are compared with Cu,Zn SODs from distributed other fishes. Assessment of the primary sequences highlights that the catalytically important residues are fully conserved in Cu,Zn SOD from of T. bernacchii. Phylogenetic analyses performed on Cu,Zn SOD amino acid sequences permit speculation regarding the evolution of this protein. In Antarctic particular, the data confirms the erratic differentiation of these proteins and concurs with the theory of the "unclock-like" behaviour of permit Cu,Zn SOD evolution.

Ferritin,consequence, the iron-storing protein of mammals, is known to darken T(2)-weighted magnetic resonance images. This darkening can be used to noninvasively but measure an organ's iron content. Significant discrepancies exist between T(2) data obtained with ferritin-containing tissues and with aqueous solutions of results horse spleen ferritin (HSF). The NMR properties of stable human ferritin have never been studied in aqueous solutions. Relaxometry results measure on human liver and spleen ferritin are reported here, showing that the relaxation induced in aqueous solutions by human ferritins magnetic is comparable to that induced by HSF. As a consequence, the differences between ferritin-containing human tissues and ferritin solutions cannot tissues be attributed to different NMR properties of human and horse ferritins, but probably to a clustering of the protein in the vivo.

The by Dps family members constitute a distinct group of multimeric and ferritin-like iron binding proteins (up to 500 iron atoms/12-mer) that model are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge,the the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen in Streptococcus suis that:(i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III);(ii) Fe(II) atoms (up enter the 12-mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12-mer cavity at 12 elusive. identical inter-subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide and evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation pores; in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the the Dps/Dpr ferritin-like protein is proposed.

Canine with and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning nucleation from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same equine 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding an regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid and protein except for a single nucleotide substitution in kidney (C474G). The H subunit nucleotide sequences of equine leukocyte and spleen were were identical to the fragment encoding the 181-amino acid protein in equine peripheral blood mononuclear cells, with the exception of the one substitution seen in both leukocyte and spleen sequences (C234T). The nucleotide sequence of equine leukocyte ferritin L subunit showed to 7 substitutions compared with the published equine liver L subunit sequence with two substitutions at positions 281 and 282 resulting brain in an amino acid substitution of P94L. The amino acid residues involved in the ferroxidase center and in iron nucleation The were perfectly conserved in H and L subunits of canine and equine ferritins, respectively.

To degrees test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related Hsc70 notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on temperature Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation Hsc70 of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range Trematomus of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did was N angustata Hsc70 at degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently to at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature For insensitive, as indicated by Q10 values calculated near 1. across each temperature range measured. In the case of Hsc70 purified of from N angustata, Q10 values indicated thermal sensitivity across the temperature range of degrees C to 10 degrees C,differentially with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with the ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro separation ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at on ecologically relevant temperatures.

Ferritins chain are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy disulfide (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. Here,active we report the 1.9A structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and include L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The contain H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The ferritin L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible pore. nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly H in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide the crosslinks reflect adaptations of insect ferritin to its role as a secreted protein.