Biomedical subjects

Domenico Bordo

Publications and source records attributed to Domenico Bordo.

12 recordsLinked to original sources

PubMed2006

Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major.

Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3(1)21/P3(2)21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 A and a = b = 111.0, c = 175.6 A, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 A.

Amino Acid Sequence↗

PubMed2006

Effects of Emx2 inactivation on the gene expression profile of neural precursors.

Emx2 plays a crucial role in the development of the diencephalon and dorsal telencephalon. Thus, Emx2-null mutants have abnormal cortical lamination and a reduction in size of the caudal and medial areas of the prosencephalon. Emx2 is expressed in neural precursors of the subventricular zone in vivo and in cultured neurospheres in vitro where it controls the size of the transit-amplifying population, affecting proliferation and clonal efficiency of neural stem cells. To identify the cellular processes mastered by Emx2, and possibly the molecular mechanisms by which the gene exerts its action, we compared the expression profile of cultured neurospheres derived from wild-type and Emx2-null mouse embryos. The differential expression of several genes was also confirmed by semiquantitative RT-PCR, real-time PCR and cytofluorimetric analysis in different preparations of neurospheres, and by in situ hybridization. The gene expression profile suggested a role for Emx2 in regulating the differentiation and migration properties of neural precursor cells. This involvement was confirmed in vitro, where the altered clonogenicity and impaired migration of Emx2-null cells were partially corrected by transduction of the Emx2 gene. Taken together, our results indicate that Emx2 is indeed involved in the transition between resident early progenitors (perhaps stem cells) and more mature precursors capable of migrating out of the ventricular zone, becoming postmitotic and differentiating into the appropriate cell type, and help explain the alterations observed in the brains of knock-out mice.

Animals↗

PubMed2004

The "rhodanese" fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli.

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic rhodaneses. In particular, conformational variation of the rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.

Amino Acid Sequence↗

PubMed2003

Expression and crystallographic characterization of the extracellular domain of human natural killer cell triggering receptor NKp46.

Human natural killer (NK) cells are regulated in their cytolytic activity by a delicate interplay between activating and inhibitory signals related to distinct families of triggering and inhibitory receptor proteins. NKp46 is a major NK cell-specific triggering receptor involved in the recognition and lysis of human and murine tumour and virally infected cells. It consists of an extracellular portion, composed of two Ig-like domains, a transmembrane segment and a small cytoplasmic domain. To shed light on the molecular-recognition events involved in NK cytotoxicity triggering mechanisms, the NKp46 extracellular region was cloned, overexpressed, refolded and crystallized. X-ray diffraction data could be collected to a resolution limit of 1.93 A. Crystals of the NKp46 extracellular region belong to the hexagonal space group P6(1) (or P6(5)), with unit-cell parameters a = b = 85.48, c = 59.91 A, gamma = 120 degrees; the asymmetric unit contains one protein chain (197 amino acids).

Amino Acid Sequence↗

PubMed2003

Structure of the human NK cell triggering receptor NKp46 ectodomain.

NKp46, a natural killer (NK) cell-specific receptor, has been recently identified as one of the triggering receptors involved in NK cell activation mediated by non-HLA class I ligands. The structure of the NKp46 extracellular receptor region, here reported, consists of two Ig-like domains assembled similarly to leukocyte immunoglobulin-like receptors (LIRs) and killer inhibitory receptors (KIRs). The extensive NKp46 residue substitutions at sites structurally related to those mediating interaction with HLA antigens in LIRs and KIRs indicate that NKp46 recognition processes in vivo should involve non-HLA ligands. NKP46 is shown to stem from an ancestral KIR/LIR family. However, the absence of close paralogues, such as those found for LIR and KIR, indicates that NKp46 is the unique member of a distinct Ig-like subfamily and suggests a specific role, which appears to be maintained across primates and rodents.

Amino Acid Sequence↗

PubMed2003

Inhibition of Azotobacter vinelandii rhodanese by NO-donors.

Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), (+/-)E-4-ethyl-2-[E-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetyl-penicillamine (SNAP) on the catalytic activity of Azotobacter vinelandii rhodanese (RhdA) has been investigated. GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion. The absorption spectrum of the NOR-3-treated RhdA displays a maximum at 335 nm, indicating NO-mediated S-nitrosylation. RhdA inhibition by NO-donors correlates with S-nitrosothiol formation. The reducing agent dithiothreitol prevents RhdA inhibition by NO-donors, fully restores the catalytic activity, and reverts the NOR-3-induced RhdA absorption spectrum to that of the active enzyme. These results indicate that RhdA inhibition occurs via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue.

Azotobacter vinelandii↗

PubMed2003

The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity.

Natural killer (NK) cells direct cytotoxicity against tumor or virally infected cells. NK cell activation depends on a fine balance between inhibitory and activating receptors. NKp44 is a cytotoxicity activating receptor composed of one Ig-like extracellular domain, a transmembrane segment, and a cytoplasmic domain. The 2.2 A crystal structure shows that the NKp44 Ig domain forms a saddle-shaped dimer, where a charged surface groove protrudes from the core structure in each subunit. NKp44 Ig domain disulfide bridge topology defines a new Ig structural subfamily. The data presented are a first step toward understanding the molecular basis for ligand recognition by natural cytotoxicity receptors, whose key role in the immune system is established, but whose cellular ligands are still elusive.

Amino Acid Sequence↗

PubMed2003

An Italian dominant FALS Leu144Phe SOD1 mutation: genotype-phenotype correlation.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease. Mutations of the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of autosomal dominant familial ALS (FALS). RESULTS: We examined the clinical features of the first Italian FALS with the Leu144Phe SOD1 mutation. Seven affected members were identified in a six-generation pedigree. A slowly progressive course (20.4+/-14.6 years) was observed in five patients. One patient died of cardiac failure two years after the onset of the disease. The propositus is still alive. Neurological manifestations began in the legs in all patients, while bulbar signs were absent or appeared late in the course of the disease. DISCUSSION: There is evidence of a correlation between this mutation and a slowly progressive phenotype of ALS. Moreover this rare mutation might derive from a common ancestor.

Adolescent↗

PubMed2002

SseA, a 3-mercaptopyruvate sulfurtransferase from Escherichia coli: crystallization and preliminary crystallographic data.

SseA, the translation product of the Escherichia coli sseA gene, is a 31 kDa protein endowed with 3-mercaptopyruvate:cyanide sulfurtransferase activity in vitro. As such, SseA is the prototype of a sulfurtransferase subfamily distinguished from the better known rhodanese sulfurtransferases, which display thiosulfate:cyanide sulfurtransferase activity. The physiological role of the two homologous enzyme families, whose catalytic activity is centred on a reactive invariant cysteine, is a matter of debate. In this framework, the forthcoming crystal structure analysis of SseA will be based on the tetragonal crystal form (space group P4(1) or P4(3)) reported here, with unit-cell parameters a = b = 150.2, c = 37.9 A.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary crystallographic characterization of the extracellular Ig-like domain of human natural killer cell activating receptor NKp44.

The cytolytic activity of human natural killer (NK) cells is regulated by fine balancing between activating and inhibitory signals related to distinct families of inhibitory and triggering receptor molecules. The recent identification of NKp44, a triggering receptor selectively expressed by activated NK cells and playing an important role in the natural cytotoxicity of these cells, opens the way to the characterization of the mechanisms involved in NKp44-mediated NK-cell activation on a structural basis. For this purpose, the NKp44 extracellular Ig-like domain was cloned, overexpressed, refolded and crystallized. Diffraction intensities were collected to a resolution limit of 2.2 A on a synchrotron source. NKp44 extracellular Ig-like domain crystals belong to the hexagonal space group P6(2)22 (or P6(4)22), with unit-cell parameters a = b = 60.4, c = 197.2 A, gamma = 120 degrees. Evaluation of the crystal packing parameter suggests the presence of one molecule (13.8 kDa) per asymmetric unit, with a solvent volume of 67%.

Amino Acid Sequence↗

PubMed2002

The rhodanese/Cdc25 phosphatase superfamily. Sequence-structure-function relations.

Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. They are found as tandem repeats, with the C-terminal domain hosting the properly structured active-site Cys residue, as single domain proteins or in combination with distinct protein domains. An increasing number of reports indicate that rhodanese modules are versatile sulfur carriers that have adapted their function to fulfill the need for reactive sulfane sulfur in distinct metabolic and regulatory pathways. Recent investigations have shown that rhodanese domains are also structurally related to the catalytic subunit of Cdc25 phosphatase enzymes and that the two enzyme families are likely to share a common evolutionary origin. In this review, the rhodanese/Cdc25 phosphatase superfamily is analyzed. Although the identification of their biological substrates has thus far proven elusive, the emerging picture points to a role for the amino-acid composition of the active-site loop in substrate recognition/specificity. Furthermore, the frequently observed association of catalytically inactive rhodanese modules with other protein domains suggests a distinct regulatory role for these inactive domains, possibly in connection with signaling.

Animals↗

PubMed2001

Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene.

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "Döhle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.

Amino Acid Sequence↗