Mitochondrial cytochromes c: a comparative analysis

c proteins of known sequence have been modeled in the oxidized state based on the existing crystallographic and NMR structures. The secondary structural elements and the overall three-dimensional structure were found to be maintained throughout the superfamily, despite variability in the sequence of individual proteins. The iron axial ligands and their reciprocal orientation were found to be nearly universally conserved. Residues constituting the hydrophobic core of the protein are also very highly conserved or conservatively substituted. Certain surface-exposed charged as well as hydrophobic groups have also been found to be conserved to the same degree as core residues. Patterns of conservation of exposed residues identify regions of the protein that are likely to be critical for its function in electron transfer. Received: 4 June 1999 / Accepted: 28 September 1999     

Incorporation and intraparticulate hydrolysis of 131 I-albumin by liver and kidney of an amphibian (Bufo arenarum)

After a single injection of formaldehyde-treated 131 I-albumin into the heart, the incorporation of the labelled protein by liver (% of total injected radioactivity/% of body weight of the organ) was far greater than in other organs. In kidney and spleen it was respectively six and three times greater than in lungs, intestine, testis and fatty body. No radioactivity was found in brain. The radioactivity in liver and kidney reached a peak 30 minutes after the injection, and quickly decreased during the following four hours. In the 27,000 g × ten minute particles recovered from liver homogenates of animals sacrificed at various times after injection, the rate of 131 I-albumin hydrolysis in vitro and the percentage of trichloroacetic acid soluble radioactivity at zero time of incubation showed different stages of intraparticulate hydrolytic activity. The incorporation and intraparticulate hydrolysis in toad kidney was very low if compared with that of toad liver or mouse kidney; however the catheptic specific activity in toad kidney doubles that of mouse kidney. Isolated toad liver was perfused with total blood, containing 131 I-albumin, for five hours at 22°C in a special chamber. In this conditions, 16% of the labelled albumin was hydrolyzed by the liver.     

Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204.     

Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed. Proteins 29:68–76, 1997. © 1997 Wiley-Liss, Inc.     

Sulfonamido-derivatives of unsubstituted carbazoles as BACE1 inhibitors

A novel series of variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylsulfonamides has been synthesized and assayed for β-Secretase (BACE1) inhibitory activity. BACE1 is a widely recognized drug target for the prevention and treatment of Alzheimer's Disease (AD). The introduction of benzyl substituents on the nitrogen atom of the arylsulfonamide moiety has so far led to the best results, with three derivatives showing IC₅₀ values ranging from 1.6 to 1.9?μM. Therefore, a significant improvement over the previously reported series of N-carboxamides (displaying IC₅₀’s?≥?2.5?μM) has been achieved, thus suggesting an active role of the sulfonamido-portion in the inhibition process. Preliminary molecular modeling studies have been carried out to rationalize the observed structure-activity relationships.     

A structural and dynamic characterization of the EF-hand protein CLSP

The structure and dynamics of human calmodulin-like skin protein (CLSP) have been characterized by NMR spectroscopy. The mobility of CLSP has been found to be different for the N-terminal and C-terminal domains. The isolated domains were also expressed and analyzed. The structure of the isolated C-terminal domain is presented. The N-terminal domain is characterized by four stable helices, which experience large fluctuations. This is shown to be due to mutations in the hydrophobic core. The overall N-terminal domain behavior is similar both in the full-length protein and in the isolated domain. By exploiting the capability of Tb3+ bound to CLSP to induce partial orientation of the molecule in a magnetic field, restricted motion of one domain with respect to the other was proved. By using NMR, ITC, and ESI-MS, the calcium and magnesium binding properties were investigated. Finally, CLSP is framed into the evolutionary scheme of the calmodulin-like family.     

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.     

Effects of age, gender and time on receptor expression and anti-Aspergillus functions of human phagocytes

Phagocytes play a central role in immune defense. Their dysfunction predisposes to infections. This study determined the expression level of nine receptors involved in Aspergillus immune response as well as the values of phagocytosis and production of radical oxygen species after Aspergillus stimulation, in a healthy adult population.The expression values of the CD11b, CD11c, CD14, CD18, CD35, CD181, CD182, CD282 and CD284 receptors on peripheral human monocytes and granulocytes was established. A heterogenous expression of the CD282 on granulocytes was observed as CD181, CD182 and CD284 on monocytes. Similarly, we observed considerable variation in the expression of these receptors over time. Only CD282 on granulocytes varied with sex. No variation with age was observed. Adherence of Aspergillus conidia to phagocytes was dependent of individual, sex, age and time. A better characterization of these innate immunity parameters is necessary to develop in the future an immunologic surveillance strategy for transplant recipients.     

A docking approach to the study of copper trafficking proteins; interaction between metallochaperones and soluble domains of copper ATPases

A structural model of the transient complex between the yeast copper chaperone Atx1 and the first soluble domain of the copper transporting ATPase Ccc2 was obtained with HADDOCK, combining NMR chemical shift mapping information with in silico docking. These two proteins are involved in copper trafficking in yeast cells. Calculations were performed starting with the copper ion either bound to Atx1 or to Ccc2 and using the experimental structures of the copper-loaded and apo forms of each protein. The copper binding motifs of the two proteins are found in close proximity. Copper tends to move from Atx1 to Ccc2, consistent with the physiological direction of transfer, with concomitant structural rearrangements, in agreement with experimental observations. The interaction is mainly of an electrostatic nature with hydrogen bonds stabilizing the complex. The structural data are relevant for a number of proteins homologous to Atx1 and Ccc2 and conserved from bacteria to humans.