Structure-function relationships in heme-proteins

Biological systems rely on heme-proteins to carry out a number of basic functions essential for their survival. Hemes, or iron-porphyrin complexes, are the versatile and ubiquitous active centers of these proteins. In the past decade, discovery of new heme-proteins, together with functional and structural research, provided a wealth of information on these diverse and biologically important molecules. Structure determination work has shown that nature has used a variety of different scaffolds and architectures to bind heme and modulate functions such as redox properties. Structural data have also provided insights into the heme-linked protein conformational changes required in many regulatory heme-proteins. Remarkable efforts have been made towards the understanding of factors governing redox potentials. Site-directed mutagenesis studies and theoretical calculations on heme environments investigated the roles of hydrophobic and electrostatic residues, and analyzed the effect of heme solvent accessibility. This review focuses on the structure-function relationships underlying the association of heme in signaling and iron metabolism proteins. In addition, an account is given about molecular features affecting heme's redox properties; this briefly revisits previous conclusions in the light of some more recent reports.     

Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching

Kallmann syndrome is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of Kallmann syndrome, KAL-1, encodes a secreted protein of still elusive function. It has been proposed that KAL-1 might be involved in some aspects of olfactory axon guidance. However, the unavailability of a mouse model, and the difficulties in studying cellular and axonal migration in vertebrates have hampered an understanding of its function. We have identified the C. elegans homolog, kal-1, and document its function in vivo. We show that kal-1 is part of a mechanism by which neurons influence migration and adhesion of epidermal cells undergoing morphogenesis during ventral enclosure and male tail formation. We also show that kal-1 affects neurite outgrowth in vivo by modulating branching. Finally, we find that human KAL-1 cDNA can compensate for the loss of worm kal-1 and that overexpression of worm or human KAL-1 cDNAs in the nematode results in the same phenotypes. These data indicate functional conservation between the human and nematode proteins and establish C. elegans as a powerful animal in which to investigate KAL function in vivo. Our findings add a new player to the set of molecules, which appear to underlie both morphogenesis and axonal/neuronal navigation in vertebrates and invertebrates.     

Inhibition of acetylcholinesterase by physostigmine analogs: conformational mobility of cysteine loop due to the steric effect of the alkyl chain

The effect of a series of physostigmine analogs on acetylcholinesterase activity was investigated. The second-order rate constant k(on) of the enzyme-inhibitor complex correlates with the conformational positioning of aromatic residues, especially Trp84, in the transition state complex. The van der Waals interactions are an important structural element of this conformational change. A transient mobility of the cysteine loop (Cys67-Cys94) was confined only to the presence of a significant steric effect. Even with this limitation, however, the steric effect seems to be an appropriate model for future tests on the "back door" hypothesis involving facilitated opening for faster product clearance.     

SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D

A naturally arising point mutation in the env gene of HIV-1 activates the aberrant inclusion of the cryptic exon 6D into most viral messages, leading to inefficient viral replication. We set out to understand how a single nucleotide substitution could cause such a dramatic change in splicing. We have determined that the exon 6D mutation promotes binding of the SR protein SC35 to the exon. Mutant exon 6D sequences function as a splicing enhancer when inserted into an enhancer-dependent splicing construct. hnRNP H family proteins bind to the enhancer as well; their binding is dependent on the sequence GGGA located just downstream of the point mutation and depletion-- reconstitution studies show that hnRNP H is essential for enhancer activity. A polypurine sequence located further downstream in exon 6D binds SR proteins but acts as an exonic splicing silencer. hnRNP H is required for interaction of U1 snRNP with the enhancer, independent of the point mutation. We propose that SC35 binding to the point mutation region may convert the hnRNP H-U1 snRNP complex into a splicing enhancer.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.     

Amyloid-β Deposition in Alzheimer Transgenic Mice Is Associated with Oxidative Stress

Abstract: Increased awareness for a role of oxidative stress in the pathogenesis of Alzheimer's disease has highlighted the issue of whether oxidative damage is a fundamental step in the pathogenesis or instead results from disease-associated pathology. In vitro experiments support both possibilities: Oxidative stress increases amyloid-β production, and, conversely, amyloid-β increases oxidative damage. To address the relationship between amyloid-β and oxidative stress in vivo, we examined, using an array of oxidative markers, transgenic mice that overexpress amyloid-β precursor protein and, as in Alzheimer's disease, develop characteristic amyloid-β deposits within the brain parenchyma. Transgenic animals show the same type of oxidative damage that is found in Alzheimer's disease, and it is important that this damage directly correlates with the presence of amyloid-β deposits. The significance of these studies is twofold. First, they provide evidence that amyloid-β and oxidative damage are inextricably linked in vivo. Second, they support the use of transgenic animals for the development of antioxidant therapeutic strategies.