Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin

Evidence of destabilization of the human thymidylate synthase (hTS) dimeric structure induced by the interface mutation Q62R

In human cells, thymidylate synthase (TS) provides the only source of 2\u2019-deoxythymidyne-5\u2019-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors

A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases

The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases. (c) 2005 Elsevier Ltd. All rights reserved

Primary health care in rural Malawi - a qualitative assessment exploring the relevance of the community-directed interventions approach

BACKGROUND: Primary Health Care (PHC) is a strategy endorsed for attaining equitable access to basic health care including treatment and prevention of endemic diseases. Thirty four years later, its implementation remains sub-optimal in most Sub-Saharan African countries that access to health interventions is still a major challenge for a large proportion of the rural population. Community-directed treatment with ivermectin (CDTi) and community-directed interventions (CDI) are participatory approaches to strengthen health care at community level. Both approaches are based on values and principles associated with PHC. The CDI approach has successfully been used to improve the delivery of interventions in areas that have previously used CDTi. However, little is known about the added value of community participation in areas without prior experience with CDTi. This study aimed at assessing PHC in two rural Malawian districts without CDTi experience with a view to explore the relevance of the CDI approach. We examined health service providers’ and beneficiaries’ perceptions on existing PHC practices, and their perspectives on official priorities and strategies to strengthen PHC. METHODS: We conducted 27 key informant interviews with health officials and partners at national, district and health centre levels; 32 focus group discussions with community members and in-depth interviews with 32 community members and 32 community leaders. Additionally, official PHC related documents were reviewed. RESULTS: The findings show that there is a functional PHC system in place in the two study districts, though its implementation is faced with various challenges related to accessibility of services and shortage of resources. Health service providers and consumers shared perceptions on the importance of intensifying community participation to strengthen PHC, particularly within the areas of provision of insecticide treated bed nets, home case management for malaria, management of diarrhoeal diseases, treatment of schistosomiasis and provision of food supplements against malnutrition. CONCLUSION: Our study indicates that intensified community participation based on the CDI approach can be considered as a realistic means to increase accessibility of certain vital interventions at community level