Toward engineering the stability and hemin-binding properties of microsomal cytochromes b5 into rat outer mitochondrial membrane cytochrome b5: examining the influence of residues 25 and 71

As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Mc) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b(5) were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Mc cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/I25L/I32L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Mc isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > I25L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Mc isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Mc cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Mc and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Leu-71 plays a pivotal role in the stabilization of rat OM apocyt b(5), presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 71, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Mc cyt b(5). During the course of these investigations we also discovered that rat OM cyt b(5) can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.     

Effect of Calcifications on Breast Ultrasound Shear Wave Elastography: An Investigational Study

PurposeTo investigate the effects of macrocalcifications and clustered microcalcifications associated with benign breast masses on shear wave elastography (SWE).MethodsSuperSonic Imagine (SSI) and comb-push ultrasound shear elastography (CUSE) were performed on three sets of phantoms to investigate how calcifications of different sizes and distributions influence measured elasticity. To demonstrate the effect in vivo, three female patients with benign breast masses associated with mammographically-identified calcifications were evaluated by CUSE.ResultsApparent maximum elasticity (E_max) estimates resulting from individual macrocalcifications (with diameters of 2mm, 3mm, 5mm, 6mm, 9mm, 11mm, and 15mm) showed values over 50 kPa for all cases, which represents more than 100% increase over background (~21kPa). We considered a 2cm-diameter circular region of interest for all phantom experiments. Mean elasticity (E_mean) values varied from 26 kPa to 73 kPa, depending on the macrocalcification size. Highly dense clusters of microcalcifications showed higher E_max values than clusters of microcalcification with low concentrations, but the difference in E_mean values was not significant.ConclusionsOur results demonstrate that the presence of large isolated macrocalcifications and highly concentrated clusters of microcalcifications can introduce areas with apparent high elasticity in SWE. Considering that benign breast masses normally have significantly lower elasticity values than malignant tumors, such areas with high elasticity appearing due to presence of calcification in benign breast masses may lead to misdiagnosis.     

Silver‐induced conformational changes of polypeptides: a CD study

The role of silver ions in various pathologies, as well as their effect on peptide conformation and properties are less understood. Consequently, we synthesized several peptides with various residues in their sequence to investigate silver‐induced conformational changes at various pH values by Circular Dichroism spectroscopy. Uniquely, the glycine‐based, histidine‐containing peptide showed a severe change from a random coil and β‐turn conformation to large α‐helices during silver binding. When comparing the effect of silver ions on the conformation of bradykinin a similar tendency was found. Besides, silver ions reduced the amyloid‐β peptide tendency to aggregation. Our results suggest a specific and protective role for silver ions in brain pathologies, which is related to their high affinity toward physiologically and pharmacologically active peptides. Fourier transform infrared spectroscopy studies as well as the mass spectrometric ones support our conclusions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Cadmium Accumulation and Tolerance in <Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. (Peanut Microsymbionts)

In this study, the effect of cadmium (Cd) on cell viability and its accumulation in Bradyrhizobium spp. (peanut microsymbionts) as well as the role of glutathione (GSH) in the tolerance to this metal were investigated. A reference strain recommended as peanut inoculant (Bradyrhizobium sp. SEMIA6144) grew up to 10 μM Cd meanwhile a GSH-deficient mutant strain (Bradyrhizobium sp. SEMIA6144-S7Z) was unable to grow at this concentration. Two native peanut isolates obtained from Córdoba soils (Bradyrhizobium sp. NLH25 and Bradyrhizobium sp. NOD31) tolerated up to 30 μM Cd. The analysis of Cd content showed that Bradyrhizobium sp. SEMIA6144 accumulated a high amount of this metal, but a considerable inhibition of growth was observed compared to tolerant strains at 10 μM Cd. At this concentration, the intracellular GSH content of all the Bradyrhizobium sp. strains was not modified in comparison to control conditions. However, at 30 μM Cd, the intracellular GSH content significantly increased in Bradyrhizobium sp. strains NLH25 and NOD31. Thus, the distinct response of each Bradyrhizobium sp. strain to Cd reveals that, even in closely related lineages, there are strain-specific variations influencing the levels of tolerance to this metal. Indeed, the native peanut isolates tolerated higher Cd concentration than the reference strain, possibly due to an increase in GSH levels which could act as a detoxifying agent.     

Carboxydipeptidase activities of recombinant cysteine peptidases. Cruzain of Trypanosoma cruzi and CPB of Leishmania mexicana.

The recombinant cysteine peptidases, cruzain from Trypanosoma cruzi and CPB2.8DeltaCTE from Leishmania mexicana, are cathepsin L-like and characteristically endopeptidases. In this study, we characterized the carboxydipeptidase activities of these enzymes and compared them with those of human recombinant cathepsin B and cathepsin L. The analysis used the internally quenched fluorescent peptide Abz-FRFK*-OH and some of its analogues, where Abz is ortho-aminobenzoic acid and K* is (2,4-dinitrophenyl)-epsilon-NH2-lysine. These peptides were demonstrated to be very sensitive substrates, due to the strong quenching effect of K* on the fluorescence of the Abz group. The carboxydipeptidase activity of cruzain was shown to be very similar to that of cathepsin B, while that of CPB2.8DeltaCTE is closer to the carboxydipeptidase activity of cathepsin L. The S2 subsite architecture of cruzain and the nature of the amino acid at the P2 position of the substrates determine its carboxydipeptidase activity and gives further and direct support to the notion that the carboxydipeptidase activity of the papain family cysteine peptidases rely on the S2-P2 interaction [N?gler D. K., Tam, W., Storer, A.C., Krupa, J.C., Mort, J.S. & Menard, R. (1999) Biochemistry38, 4868-4874]. Cruzain and CPB2.8DeltaCTE presented a broad pH-range for both the endo- and exo-peptidase activities, although the later is approximately one order of magnitude lower. This feature, that is not common in related mammalian cysteine peptidases, is consistent with the enzymes being exposed to different environmental conditions and having different locations during parasite development.