Pathogenic mechanism and modeling of neuroferritinopathy.

Neuroferritinopathy is a rare autosomal dominant inherited movement disorder caused by alteration of the L-ferritin gene that results in the production of a ferritin molecule that is unable to properly manage iron, leading to the presence of free redox-active iron in the cytosol. This form of iron has detrimental effects on cells, particularly severe for neuronal cells, which are highly sensitive to oxidative stress. Although very rare, the disorder is notable for two reasons. First, neuroferritinopathy displays features also found in a larger group of disorders named Neurodegeneration with Brain Iron Accumulation (NBIA), such as iron deposition in the basal ganglia and extrapyramidal symptoms; thus, the elucidation of its pathogenic mechanism may contribute to clarifying the incompletely understood aspects of NBIA. Second, neuroferritinopathy shows the characteristic signs of an accelerated process of aging; thus, it can be considered an interesting model to study the progress of aging. Here, we will review the clinical and neurological features of neuroferritinopathy and summarize biochemical studies and data from cellular and animal models to propose a pathogenic mechanism of the disorder.

Stem Cell Modeling of Neuroferritinopathy Reveals Iron as a Determinant of Senescence and Ferroptosis during Neuronal Aging.

Neuroferritinopathy (NF) is a movement disorder caused by alterations in the L-ferritin gene that generate cytosolic free iron. NF is a unique pathophysiological model for determining the direct consequences of cell iron dysregulation. We established lines of induced pluripotent stem cells from fibroblasts from two NF patients and one isogenic control obtained by CRISPR/Cas9 technology. NF fibroblasts, neural progenitors, and neurons exhibited the presence of increased cytosolic iron, which was also detectable as: ferritin aggregates, alterations in the iron parameters, oxidative damage, and the onset of a senescence phenotype, particularly severe in the neurons. In this spontaneous senescence model, NF cells had impaired survival and died by ferroptosis. Thus, non-ferritin-bound iron is sufficient per se to cause both cell senescence and ferroptotic cell death in human fibroblasts and neurons. These results provide strong evidence supporting the primary role of iron in neuronal aging and degeneration.

Neuroferritinopathy: From ferritin structure modification to pathogenetic mechanism.

Neuroferritinopathy is a rare, late-onset, dominantly inherited movement disorder caused by mutations in L-ferritin gene. It is characterized by iron and ferritin aggregate accumulation in brain, normal or low serum ferritin levels and high variable clinical feature. To date, nine causative mutations have been identified and eight of them are frameshift mutations determined by nucleotide(s) insertion in the exon 4 of L-ferritin gene altering the structural conformation of the C-terminus of the L-ferritin subunit. Acting in a dominant negative manner, mutations are responsible for an impairment of the iron storage efficiency of ferritin molecule. Here, we review the main characteristics of neuroferritinopathy and present a computational analysis of some representative recently defined mutations with the purpose to gain new information about the pathogenetic mechanism of the disorder. This is particularly important as neuroferritinopathy can be considered an interesting model to study the relationship between iron, oxidative stress and neurodegeneration.

Molecular dynamics and docking simulation of a natural variant of Activated Protein C with impaired protease activity: implications for integrin-mediated antiseptic function.

Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVß3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.

BRCA1 point mutations in premenopausal breast cancer patients from Central Sudan.

Premenopausal breast cancer (BC) is one of the most common cancers of women in rural Africa and part of the disease load may be related to hereditary predisposition, including mutations in the BRCA1 gene. However, the BRCA1 mutations associated with BC in Africa are scarcely characterized. We report here 33 BRCA1 point mutations, among which 2 novel missense variants, found in 59 Central Sudanese premenopausal BC patients. The high fractions of mutations with intercontinental and uniquely African distribution (17/33, 51.5 % and 14/33, 42.4 %, respectively) are in agreement with the high genetic diversity expected in an African population. Overall 24/33 variants (72.7 %) resulted neutral; 8/33 of unknown significance (24.3 %, including the 2 novel missense mutations); 1 (3.0 %) overtly deleterious. Notably, in silico studies predict that the novel C-terminal missense variant c.5090G>A (p.Cys1697Tyr) affects phosphopeptide recognition by the BRCA1 BRCT1 domain and may have a pathogenic impact. Genetic variation and frequency of unique or rare mutations of uncertain clinical relevance pose significant challenges to BRCA1 testing in Sudan, as it might happen in other low-resource rural African contexts.

Study of FTMT and ABCA4 genes in a patient affected by age-related macular degeneration: identification and analysis of new mutations.

BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial disease for which an involvement of alterations in the retinal ABC transporter gene (ABCA4) is still debated. Oxidative stress in retinal pigment epithelial cells has been postulated to contribute to the pathogenesis of the disease. Mitochondrial ferritin (FtMt), an iron-sequestering protein, is expressed in cell types characterized by high metabolic activity and oxygen consumption, including human retina, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. Based on these findings we wanted to investigate whether mutations in this gene could be found in AMD patients. METHODS: Mutational scanning of the FTMTgene was performed in a cohort of 50 patients affected by age-related macular degeneration. The ABCA4 gene was also scanned in one patient carrying an FtMt mutation. In silico analyses were carried out on the identified variants. The recombinant form of FtMt variant was expressed in Escherichia coli and biochemically characterized. RESULTS: One patient was found to be heterozygous for two previously unreported genetic changes: a complex FtMt mutation (c.437_450delinsCT: delAGGACATCAAGAAGinsCT) and a missense p.Leu973Phe (c.2919G>T) mutation in exon 20 of ABCA4. Computational analyses predicted a severe structural impairment for FtMt variant and a mild destabilizing effect for ABCA4. E. coli expression of recombinant FtMt variant yielded a highly insoluble protein that could not be renatured under in vitro conditions suitable for wild-type ferritins. CONCLUSIONS: Our findings suggest that the FtMt mutation may determine a condition similar to haploinsufficiency resulting in a reduced protection from iron-dependent oxidative stress in mitochondria.

Molecular chaperones as therapeutic targets to counteract proteostasis defects.

The health of cells is preserved by the levels and correct folding states of the proteome, which is generated and maintained by the proteostasis network, an integrated biological system consisting of several cytoprotective and degradative pathways. Indeed, the health conditions of the proteostasis network is a fundamental prerequisite to life as the inability to cope with the mismanagement of protein folding arising from genetic, epigenetic, and micro-environment stress appears to trigger a whole spectrum of unrelated diseases. Here we describe the potential functional role of the proteostasis network in tumor biology and in conformational diseases debating on how the signaling branches of this biological system may be manipulated to develop more efficacious and selective therapeutic strategies. We discuss the dual strategy of these processes in modulating the folding activity of molecular chaperones in order to counteract the antithetic proteostasis deficiencies occurring in cancer and loss/gain of function diseases. Finally, we provide perspectives on how to improve the outcome of these disorders by taking advantage of proteostasis modeling.

Exploring the role of the phospholipid ligand in endothelial protein C receptor: a molecular dynamics study.

Endothelial protein C receptor (EPCR) is a CD1-like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti-inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen-presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC gamma-carboxyglutamic acid (Gla) domain binding. However, the lipid-free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand-free conformations. Compared with the bound form, unbound structures showed a narrowing of the A' pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution-induced conformational changes affecting helices around the A' pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests.

In silico saturation mutagenesis and docking screening for the analysis of protein-ligand interaction: the Endothelial Protein C Receptor case study.

BACKGROUND: The design of mutants in protein functional regions, such as the ligand binding sites, is a powerful approach to recognize the determinants of specific protein activities in cellular pathways. For an exhaustive analysis of selected positions of protein structure large scale mutagenesis techniques are often employed, with laborious and time consuming experimental set-up. 'In silico' mutagenesis and screening simulation represents a valid alternative to laboratory methods to drive the 'in vivo' testing toward more focused objectives. RESULTS: We present here a high performance computational procedure for large-scale mutant modelling and subsequent evaluation of the effect on ligand binding affinity. The mutagenesis was performed with a 'saturation' approach, where all 20 natural amino acids were tested in positions involved in ligand binding sites. Each modelled mutant was subjected to molecular docking simulation and stability evaluation. The simulated protein-ligand complexes were screened for their impairment of binding ability based on change of calculated Ki compared to the wild-type.An example of application to the Endothelial Protein C Receptor residues involved in lipid binding is reported. CONCLUSION: The computational pipeline presented in this work is a useful tool for the design of structurally stable mutants with altered affinity for ligand binding, considerably reducing the number of mutants to be experimentally tested. The saturation mutagenesis procedure does not require previous knowledge of functional role of the residues involved and allows extensive exploration of all possible substitutions and their pairwise combinations. Mutants are screened by docking simulation and stability evaluation followed by a rationally driven selection of those presenting the required characteristics. The method can be employed in molecular recognition studies and as a preliminary approach to select models for experimental testing.

Virtual screening pipeline and ligand modelling for H5N1 neuraminidase.

The H5N1 virus neuraminidase structure was solved in two different conformations depending on the inhibitor concentration. In the absence of oseltamivir or at a low concentration, the neuraminidase structure assumes an open form that closes at a high oseltamivir concentration due to the shift of the so-called 150-loop near the active site. Although the close conformation is similar to all the other structurally known neuraminidase types, it doesn't appear to be the most likely physiological condition for N1. To investigate the specific ligand binding properties of the open form, we screened by docking simulation, a large dataset of ligands and compared the results with closed form. The virtual screening procedure was implemented in a docking pipeline that also performs a step-by-step, target specific, filtering approach for data reduction. The selected ligands display binding ability involving multiple sites of interaction including the active site and an adjacent cavity made available by the 150-loop shift. Two ligands are especially interesting and are proposed as substituents to design oseltamivir derivatives specifically suited for the open conformation.

The role of iron in mitochondrial function.

BACKGROUND: Iron is an essential element for life, as it is a cofactor for enzymes involved in many metabolic processes, but it can also be harmful, since its excess is thought to enhance the production of reactive oxygen species and induce oxidative damage. Iron is transformed into its biologically available form in the mitochondrion by the iron-sulfur (Fe/S) cluster and heme synthesis pathways. During the past decade, substantial progress has been made in the elucidation of iron-linked mechanisms that occur in the mitochondrion, demonstrating the crucial role played by this organelle in maintaining cellular iron homeostasis. GENERAL SIGNIFICANCE: This review summarizes current knowledge of the mechanisms underlying iron trafficking in mitochondria and how it is handled inside the organelle. Relevant updates with regard to the Fe/S cluster and heme biosynthetic pathways, as well as the relationship between mitochondrial iron homeostasis impairment and related diseases, are also discussed.

ProCMD: a database and 3D web resource for protein C mutants.

BACKGROUND: Activated Protein C (ProC) is an anticoagulant plasma serine protease which also plays an important role in controlling inflammation and cell proliferation. Several mutations of the gene are associated with phenotypic functional deficiency of protein C, and with the risk of developing venous thrombosis. Structure prediction and computational analysis of the mutants have proven to be a valuable aid in understanding the molecular aspects of clinical thrombophilia. RESULTS: We have built a specialized relational database and a search tool for natural mutants of protein C. It contains 195 entries that include 182 missense and 13 stop mutations. A menu driven search engine allows the user to retrieve stored information for each variant, that include genetic as well as structural data and a multiple alignment highlighting the substituted position. Molecular models of variants can be visualized with interactive tools; PDB coordinates of the models are also available for further analysis. Furthermore, an automatic modelling interface allows the user to generate multiple alignments and 3D models of new variants. CONCLUSION: ProCMD is an up-to-date interactive mutant database that integrates phenotypical descriptions with functional and structural data obtained by computational approaches. It will be useful in the research and clinical fields to help elucidate the chain of events leading from a molecular defect to the related disease. It is available for academics at the URL http://www.itb.cnr.it/procmd/.

Identification of two novel frameshift mutations in the KCNJ11 gene in two Italian patients affected by Congenital Hyperinsulinism of Infancy.

Congenital Hyperinsulinism of Infancy (CHI) is a genetically heterogeneous disorder characterized by profound hypoglycemia related to inappropriate insulin secretion. Two histopathologically and genetically distinct groups are recognized among patients with CHI due to ATP-sensitive potassium channel (KATP) defects: a diffuse type (Di-CHI), which involves the whole pancreas, and a focal form (Fo-CHI), which shows adenomatous islet-cell hyperplasia of a particular area within the normal pancreas. The beta-cell KATP channel consists of two essential subunits: Kir6.2 encoded by the KCNJ11 gene which is the pore-forming unit and belongs to the inwardly rectifying potassium channel family, and SUR1 (sulfonylurea receptor 1) encoded by the ABCC8 gene, which belongs to the ATP-binding cassette (ABC) transporter family. The KATP channel is an octameric complex of four Kir6.2 and four SUR1 subunits. More than one hundred mutations have been found in KATP channel genes ABCC8 and KCNJ11, but to date only twenty mutations have been identified in KCNJ11, most of them are missense mutations and only one is a single base deletion. The Fo-CHI has been demonstrated to arise in individuals who have a germline mutation in the paternal allele of ABCC8 or KCNJ11 in addition to a somatic loss of the maternally derived chromosome region 11p15 in adenomatous pancreatic beta-cells, while Di-CHI predominantly arises from the autosomal recessive inheritance of KATP channel gene mutations. Here we describe the molecular findings in nine children who presented, in the neonatal period, with signs and symptoms of hypoglycemia and diagnosed affected by CHI according to international diagnostic criteria. Direct sequencing of the complete coding exon and promoter region of KCNJ11 gene showed, in two Italian patients, two new heterozygous mutations which result in the appearance of premature translation termination codons resulting in the premature end of Kir6.2. Interestingly most of the CHI mutations detected in other population studies are situated in the ABCC8 gene.

Identification and computationally-based structural interpretation of naturally occurring variants of human protein C.

Protein C (PC) is a key regulator of blood clotting and inflammation. Its inherited deficiency is associated with venous thromboembolism, and recombinant activated PC is currently used to increase survival in severe sepsis. The molecular basis of inherited PC deficiency is heterogeneous. Due to its multiple physiologic interactions and functions, and its modular structure, natural variants aid in the understanding of the relationship between critical residues and discrete functions. This knowledge has important therapeutic implications in the planning of a recombinant activated PC with a specific therapeutic target and devoid of major collateral effects. A way of predicting important functional consequences of residue variation is the use of molecular modeling and structural interpretation of amino acidic substitutions. A study of 21 out of 32 identified PC gene (PROC) variants is presented. For three of them, localized in the active site, electrostatic potential variation was calculated. For more than half of the studied variants, an explanation for the functional impairment could be derived from computational analysis, allowing a focused choice of which variants it is worthwhile pursuing.

Modelling the interaction of steroid receptors with endocrine disrupting chemicals.

BACKGROUND: The organic polychlorinated compounds like dichlorodiphenyltrichloroethane with its metabolites and polychlorinated biphenyls are a class of highly persistent environmental contaminants. They have been recognized to have detrimental health effects both on wildlife and humans acting as endocrine disrupters due to their ability of mimicking the action of the steroid hormones, and thus interfering with hormone response. There are several experimental evidences that they bind and activate human steroid receptors. However, despite the growing concern about the toxicological activity of endocrine disrupters, molecular data of the interaction of these compounds with biological targets are still lacking. RESULTS: We have used a flexible docking approach to characterize the molecular interaction of seven endocrine disrupting chemicals with estrogen, progesterone and androgen receptors in the ligand-binding domain. All ligands docked in the buried hydrophobic cavity corresponding to the hormone steroid pocket. The interaction was characterized by multiple hydrophobic contacts involving a different number of residues facing the binding pocket, depending on ligands orientation. The EDC ligands did not display a unique binding mode, probably due to their lipophilicity and flexibility, which conferred them a great adaptability into the hydrophobic and large binding pocket of steroid receptors. CONCLUSION: Our results are in agreement with toxicological data on binding and allow to describe a pattern of interactions for a group of ECD to steroid receptors suggesting the requirement of a hydrophobic cavity to accommodate these chlorine carrying compounds. Although the affinity is lower than for hormones, their action can be brought about by a possible synergistic effect.