Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopical methods, fluorescence and circular dichroism. Three successive processes can be observed during the renaturation of this protein. First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer. Second, two monomers associate into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of ATP during renaturation increases the rate of formation of this dimeric species. The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site. The self-assembly of phosphofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.     

Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA.

Breathing 100% O2 at 1 atmosphere absolute (ATA) is known to be associated with a decrease in cerebral blood flow (CBF). It is also accompanied by a fall in arterial Pco2 leading to uncertainty as to whether the cerebral vasoconstriction is totally or only in part caused by arterial hypocapnia. We tested the hypothesis that the increase in arterial Po2 while O2 was breathed at 1.0 ATA decreases CBF independently of a concurrent fall in arterial Pco2. CBF was measured in seven healthy men aged 21-62 yr by using noninvasive continuous arterial spin-labeled-perfusion MRI. The tracer in this technique, magnetically labeled protons in blood, has a half-life of seconds, allowing repetitive measurements over short time frames without contamination. CBF and arterial blood gases were measured while breathing air, 100% O2, and 4 and 6% CO2 in air and O2 backgrounds. Arterial Po2 increased from 91.7 +/- 6.8 Torr in air to 576.7 +/- 18.9 Torr in O2. Arterial Pco2 fell from 43.3 +/- 1.8 Torr in air to 40.2 +/- 3.3 Torr in O2. CBF-arterial Pco2 response curves for the air and hyperoxic runs were nearly parallel and separated by a distance representing a 28.7-32.6% decrement in CBF. Regression analysis confirmed the independent cerebral vasoconstrictive effect of increased arterial Po2. The present results also demonstrate that the magnitude of this effect at 1.0 ATA is greater than previously measured.     

Crystallographic and spectroscopic studies of peroxide-derived myoglobin compound II and occurrence of protonated FeIV O

High resolution crystal structures of myoglobin in the pH range 5.2-8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe-O bond length (1.86-1.90 A) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe-O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an Fe(IV)-O species with a single bond. The presence of Fe(IV) is indicated by a small isomer shift of delta = 0.07 mm/s from M?ssbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.     

Receptor signaling lymphocyte-activation molecule family 1 (Slamf1) regulates membrane fusion and NADPH oxidase 2 (NOX2) activity by recruiting a Beclin-1/Vps34/ultraviolet radiation resistance-associated gene (UVRAG) complex

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor of several Gram-negative bacteria. Upon entering the phagosome of macrophages Slamf1 induces production of phosphatidylinositol 3-phosphate, which positively regulates the activity of the NOX2 enzyme and phagolysosomal maturation. Here, we report that in Escherichia coli-containing phagosomes of mouse macrophages, Slamf1 interacts with the class III PI3K Vps34 in a complex with Beclin-1 and UVRAG. Upon phagocytosis of bacteria the NOX2 activity was reduced in macrophages isolated from Beclin-1(+/-) mice compared with wild-type mice. This Slamf1/Beclin-1/Vps34/UVRAG protein complex is formed in intracellular membrane compartments as it is found without inducing phagocytosis in macrophages, human chronic lymphocytic leukemia cells, and transfectant HEK293 cells. Elimination of its cytoplasmic tail abolished the interaction of Slamf1 with the complex, but deletion or mutation of the two ITAM motifs did not. Both the BD and CCD domains of Beclin-1 were required for efficient binding to Slamf1. Because Slamf1 did not interact with Atg14L or Rubicon, which can also form a complex with Vps34 and Beclin-1, we conclude that Slamf1 recruits a subset of Vps34-associated proteins, which is involved in membrane fusion and NOX2 regulation.     

Cutting edge: Slamf8 is a negative regulator of Nox2 activity in macrophages

Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages (MΦs) by IFN-γ or bacteria. In this article, we report that a very high NADPH oxidase (Nox2) enzyme activity was found in Slamf8(-/-) MΦs in response to Escherichia coli or Staphylococcus aureus, as well as to PMA. The elevated Nox2 activity in Slamf8(-/-) MΦs was also demonstrated in E. coli or S. aureus phagosomes by using a pH indicator system and was further confirmed by a reduction in the enzyme activity after transfection of the receptor into Slamf8-deficient primary MΦs or RAW 264.7 cells. Upon exposure to bacteria or PMA, protein kinase C activity in Slamf8(-/-) MΦs is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which, in turn, leads to greater Nox2 activity. Taken together, the data show that, in response to inflammation-associated stimuli, the inducible receptor Slamf8 negatively regulates inflammatory responses.     

Ferric ion (hydr)oxo clusters in the “Venus flytrap” cleft of FbpA: M?ssbauer, calorimetric and mass spectrometric studies

Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. M?ssbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.     

Indicators of acute and persistent renal damage in adult thrombotic microangiopathy

Background

Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established.

Methods

We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency.

Results

On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097–26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400–12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death.

Conclusions

High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure.     

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.     

A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A.

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.