Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

A comparison of ceruloplasmin to biological polyanions in promoting the oxidation of Fe under physiologically relevant conditions

Background

Iron oxidation is thought to be predominantly handled enzymatically in the body, to minimize spontaneous combustion with oxygen and to facilitate cellular iron export by loading transferrin. This process may be impaired in disease, and requires more accurate analytical assays to interrogate enzymatic- and auto-oxidation within a physiologically relevant environment.

Method

A new triplex ferroxidase activity assay has been developed that overcomes the previous assay limitations of measuring iron oxidation at a physiologically relevant pH and salinity.

Results

Revised enzymatic kinetics for ceruloplasmin (V_max ≈ 35 μM Fe^3 +/min/μM; K_m ≈ 15 μM) are provided under physiological conditions, and inhibition by sodium azide (K_i for Ferric Gain 78.3 μM, K_i for transferrin loading 8.1 × 10⁴ μM) is quantified. We also used this assay to characterize the non-enzymatic oxidation of iron that proceeded linearly under physiological conditions.

Conclusions and general significance

These findings indicate that the requirement of an enzyme to oxidize iron may only be necessary under conditions of adverse pH or anionic strength, for example from hypoxia. In a normal physiological environment, Fe^3 + incorporation into transferrin would be sufficiently enabled by the biological polyanions that are prevalent within extracellular fluids.     

Systemic lupus erythematosus: Molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties

Background

DNase antibodies can play an important role in the pathogenesis of different autoimmune pathologies.

Methods

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) was used. The small pools of phage particles displaying DNA binding light chains with different for DNA were isolated by affinity chromatography on DNA-cellulose and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Forty-five of 451 individual colonies were randomly chosen for a study of MLChs with DNase activity. The clones were expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography followed by gel filtration, and studied in detail.

Results

Fifteen of 45 MLChs efficiently hydrolyzed DNA, and fourteen of them demonstrated various optimal concentrations of KCl or NaCl in a 1–100 mM range and showed one or two pH optima in a 4.8–9.1 range. All MLChs were dependent on divalent metal cations: the ratio of relative DNase activity in the presence of Mn^2 +, Ca^2 +, Mg^2 +, Ni^2 +, Zn^2 +, Cu^2 +, and Co^2 + was individual for each MLCh preparation. Fourteen MLChs demonstrated a comparable affinity for DNA (260–320 nM), but different k_cat values (0.02–0.7 min^− 1).

Conclusions

These observations suggest an extreme diversity of DNase abzymes from SLE patients.

General significance

SLE light chain repertoire can serve as a source of new types of DNases.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

Aims

Accumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca^2 + aberrations.

Main methods

Adult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12 h. Contractile and intracellular Ca^2 + properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), rise in intracellular Ca^2 + Fura-2 fluorescence intensity and intracellular Ca^2 + decay. Oxidative stress, O₂⁻ production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting.

Key findings

MG-AGE compromised contractile and intracellular Ca^2 + properties including reduced PS, ± dL/dt, prolonged TPS and TR₉₀, decreased electrically stimulated rise in intracellular Ca^2 + and delayed intracellular Ca^2 + clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O₂⁻ generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP.

Significance

These findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.     

Effects of sub-chronic aluminum chloride exposure on rat ovaries

Aims

This experiment investigated the effects of sub-chronic aluminum chloride (AlCl₃) exposure on rat ovaries.

Main methods

Eighty female Wistar (5 weeks old) rats, weighed 110–120 g, were randomly divided into four treatment groups: control group (CG), low-dose group (LG, 64 mg/kg BW AlCl₃), mid-dose group (MG, 128 mg/kg BW AlCl₃) and high-dose group (HG, 256 mg/kg BW AlCl₃). The AlCl₃ was administered in drinking water for 120 days. The ovarian ultrastructure was observed. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), succinate dehydrogenase (SDH), Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Fe, Cu and Zn, and the protein expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovary were determined.

Key findings

The results showed that the structure of the ovary was disrupted, the activities of ALP, ACP, SDH, Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Zn, Fe and the protein expression of FSHR and LHR were lowered, and the content of Cu was increased in AlCl₃-treated rats than those in control.

Significance

The results indicate that sub-chronic AlCl₃ exposure caused the damage of the ovarian structure, the disturbed metabolism of Fe, Zn and Cu and the decreased activities of Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase in the ovary, which could result in suppressed energy supply in the ovary. A combination of suppression of energy supply and reduction of expression of FSHR and LHR could inhibit ovulation and corpus luteum development, leading to infertility in female rats.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression

Background

Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.

Methods

The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene.

Results

The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C₄) to laurate (C₁₂). The k_cat/K_m ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻¹, respectively.

Conclusions

The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.

General significance

The enzyme is the first characterized archaeal dienelactone hydrolase.     

Zn rather than Ca or Mg used as a cofactor in non-muscular actin from the oyster to control protein polymerization

Background

The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments (F-actin). Each actin monomer (G-actin) contains a divalent alkaline earth metal ion (in vivo Mg^2 +; in vitro usually Ca^2 +) as a cofactor that is crucial for protein polymerization. Prior to this study, however, whether or not other types of metal ions can play the same role as Mg^2 + or Ca^2 + in actins remains unknown.

Methods

A new actin from the gills of oyster (AGO) was prepared and characterized by protein purification techniques, SDS- and native-PAGE, and LC–MS\MS for the first time. The property of this protein was studied by CD, fluorescence and UV/vis spectroscopy, laser light scattering, and TEM.

Results

AGO is a monomer with a MW of ~ 42 kDa. AGO is unique among all known actins in that Zn^2 + is only a naturally binding metal in the protein, and that one native AGO molecule binds 8 zinc ions, which can be removed by EDTA treatment at pH 7.2. The presence of zinc has a great effect on the secondary and tertiary structure of the protein. Correlated with such effect is that these zinc ions in native AGO facilitate protein polymerization, whereas removal of zinc ions from native AGO results in a loss of such polymerization property.

Conclusions

The present work demonstrates that AGO is a novel zinc-binding protein with high capacity, and high selectivity.

General significance

This work extends an understanding of the function of zinc and actin.     

Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites

Background

Xyloglucan xyloglucosyl transferases (EC 2.4.1.207), known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates.

Methods

cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.

Results

Significant differences in catalytic efficiencies (k_cat·K_m⁻¹) were observed and these values were 0.01, 0.02 and 0.2 s⁻¹·mg⁻¹·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations.

Conclusions

The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.

General significance

The results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents.     

Influence of inflammatory response,infection, and pulmonary function in cystic fibrosis

Aims

Recurrent infections and activation of the inflammatory response affect the prognosis of cystic fibrosis (CF). We investigated the relationship between inflammatory response, infection, and pulmonary function in CF.

Main methods

A clinical-cross-sectional study was conducted with 86 subjects: control group (CG, n = 31, the same age and sex of the CF group), and CF group (CFG, n = 55, age: 1–16 years), further distributed into CFG negative or positive bacteriology (CFGB⁻/CFGB⁺), and CFG negative or positive Pseudomonas aeruginosa (CFGPa⁻/CFGPa⁺). Using the Wald test, multiple linear regression (95% confidence interval) was performed between CG and CFG, and between CG and each of the CF subgroups (CFGB⁻/CFGB⁺ and CFGPa⁻/CFGPa⁺). The inflammatory markers evaluated were myeloperoxidase (MPO), adenosine deaminase (ADA) activities, interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), nitric oxide metabolites (NOx) levels, and total and differential leukocyte counts.

Key findings

After adjusting for sex and age, CFG compared to CG revealed an increase of MPO, IL-1β (P < 0.001 in all subgroups), and CRP: CFG (P = 0.002), CFGB⁻ (P = 0.007), CFGB⁺ (P = 0.009), CFGPa⁻ (P = 0.004) and CFGPa⁺ (P = 0.020). NOx (P = 0.001, P < 0.001), leukocytes (P = 0.002, P = 0.001), and neutrophils (P = 0.003, P < 0.001) were increased in CFGB⁺ and CFGPa⁺, respectively. A negative correlation between FEV₁ and leukocytes (P = 0.008) and FEV₁ and neutrophils (P = 0.031) resulted in CFG.

Significance

The inflammatory response characterized by the increase of MPO, IL-1β, and CRP is determinant for CF. Also leukocytosis due to neutrophilia determines the pulmonary function deficiency in this disease.