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1.
Chem Commun (Camb) ; 53(85): 11622-11633, 2017 Oct 24.
Article in English | MEDLINE | ID: mdl-29019481

ABSTRACT

The combined use of X-ray crystallography and mass spectrometry represents a valuable strategy to investigate and characterize protein metalation induced by anticancer metal-based drugs. Here, we summarize a series of significant results recently obtained in our laboratories upon the examination of the structures of several adducts of proteins with representative metallodrugs (mostly containing ruthenium, gold and platinum). The general mechanisms of protein metalation that emerge from a careful comparative analysis of these structures are illustrated and their mechanistic implications are discussed. Possible directions for future work in the field are delineated.

2.
Biochim Biophys Acta ; 1802(6): 572-81, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20123124

ABSTRACT

We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S-S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.


Subject(s)
Dyslipidemias/genetics , Germ-Line Mutation , Intestinal Polyps/genetics , PPAR gamma/genetics , Adenocarcinoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Amino Acid Substitution , Animals , Base Sequence , Binding Sites/genetics , COS Cells , Chlorocebus aethiops , Colonic Neoplasms/genetics , DNA Primers/genetics , Dyslipidemias/metabolism , Female , Humans , In Vitro Techniques , Intestinal Polyps/metabolism , Loss of Heterozygosity , Male , Mice , Middle Aged , Models, Molecular , NIH 3T3 Cells , PPAR gamma/chemistry , PPAR gamma/metabolism , Pedigree , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection , Young Adult
3.
Xenobiotica ; 39(3): 236-48, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19280522

ABSTRACT

The metabolism of six anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans (PhEBfx) was studied in vitro using rat hepatic microsomal and cytosolic fractions as a mammalian model and whole cells of T. cruzi as a parasitic model. Some of the expected metabolites were synthesized to provide authentic chromatographic standards. The metabolites were identified using high-performance liquid chromatography (HPLC) in comparison with the authentic standards and their proportions were determined. Their structures were confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The behaviour of the six PhEBfx in the three different systems was similar. The main metabolites, formed by reductive processes, were the corresponding o-nitroanilines. Two of the test compounds were studied for extended time periods in the rat liver preparations and their terminal metabolites were identified as o-phenylendiamine derivatives.


Subject(s)
Aniline Compounds/metabolism , Benzoxazoles/metabolism , Hepatocytes/metabolism , Microsomes, Liver/metabolism , Oxadiazoles/metabolism , Trypanosoma cruzi/metabolism , Animals , Benzoxazoles/chemistry , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Mass Spectrometry , Oxadiazoles/chemistry , Rats
4.
Protein Sci ; 9(6): 1217-25, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10892814

ABSTRACT

Guanine-containing mono- and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA. 1992. J Mol Biol 224:265-267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2',5')guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 A (R-factor 0.192) and 2.0 A (R-factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.


Subject(s)
Dinucleoside Phosphates/metabolism , Ribonuclease, Pancreatic/metabolism , Crystallography, X-Ray , Models, Molecular , Protein Binding , Protein Conformation , Ribonuclease, Pancreatic/chemistry
5.
J Mol Biol ; 293(3): 569-77, 1999 Oct 29.
Article in English | MEDLINE | ID: mdl-10543951

ABSTRACT

Bovine seminal ribonuclease (BS-RNase) is a peculiar member of the pancreatic-like ribonuclease superfamily endowed with unique biological functions. It has been shown that native BS-RNase is a mixture of two distinct dimeric forms. The most abundant form is characterised by the swapping of the N-terminal helix. Kinetic studies have shown that this dimer is allosterically regulated, whereas the minor component, in which no swapping occurs, exhibits typical Michaelian kinetics. In order to correlate the catalytic properties with the structural features of BS-RNase, we have determined the crystal structure of the BS-RNase swapping dimer complexed with uridylyl(2'-5')guanosine. The structure of the complex was refined to an R value of 0.189 at 1.9 A resolution. Surprisingly, the enzyme binds four dinucleotide molecules, all in a non-productive way. In the two active sites, the guanine base is located in the subsite that is specific for pyrimidines. This unusual binding has been observed also in complexes of RNase A with guanine-containing nucleotides (retro-binding). One of the two additional dinucleotide molecules bound to the enzyme is located on the surface of the protein in a pocket generated by crystal packing; the second was found in a cavity at the interface between the two subunits of the swapping dimer. There are indications that the interface site plays a role in the allosteric regulation exhibited by BS-RNase. This finding suggests that domain swapping may not merely be a mechanism that proteins adopt for the transition from a monomeric to oligomeric state but can be used to achieve modulations in catalytic function.


Subject(s)
Allosteric Site , Dinucleoside Phosphates/metabolism , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Animals , Cattle , Crystallization , Crystallography, X-Ray , Dimerization , Dinucleoside Phosphates/chemistry , Electrons , Endoribonucleases/genetics , Guanine/metabolism , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , Structure-Activity Relationship
12.
J Bone Joint Surg Am ; 55(2): 406-8, 1973 Mar.
Article in English | MEDLINE | ID: mdl-4696174
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