1,721,084 research outputs found
Applications of 1H-NMR relaxometry in the investigation of heme-human serum albumin.
No full textLondon, 2-5 July 2003
A glucosamine derivative affects metastatic activity in two prostate cancer cell lines by stimulating Maspin expression
Full text linkProstate Cancer (CaP) is the most common male tumor and is the third leading cause of cancer death, with an incidence of 1.28 million cases worldwide, according to the data collected by the Global Cancer Observatory in 2018. PC3 and LNCaP cell lines represent suitable models to study CaP development, due to their different metastatic origin and their distinct sensitiveness to androgen signaling. PC3 is a hormone-insensitive cell line isolated from a vertebral metastatic prostatic tumor. It lacks of androgen receptor (AR) and its abnormal growth could be attributed to enhanced expression levels of TGF-α, EGF, and EGF-R. LNCaP cell line was isolated from a human metastatic prostate adenocarcinoma found in a lymphnode. It expresses a T877A mutated AR form, which results in an enhanced binding affinity for several steroid compounds. Both cell lines present very low basal levels of Maspin expression. Maspin, an unusual member of the Serine Proteases superfamily, has been characterized as a class II tumor suppressor gene in many cancer types, among them CaP, due to its ability to inhibit cell invasiveness and proliferation and to increase apoptosis, thus inhibiting metastasis. In normal prostate epithelial cells, Maspin is highly expressed, whereas in prostate cancer cells its expression is almost completely suppressed. Previously in our laboratory, a glucosamine derivative, NCPA, has been proved to be effective in stimulating Maspin expression and to induce its nuclear localization in an osteosarcoma cell line, 143B. The aim of my PhD project was to evaluate the ability of NCPA to affect metastatic activity in two prostate cancer cell lines, the hormone-insensitive PC3 and the hormone-sensitive LNCaP cells, respectively representative of late-stage and early-stage CaP
Characterisation of the key enzymes involved in vitamin B6 salvage pathway in Escherichia coli and humans
Full text linkThe catalytically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), acts as a coenzyme in a variety of different enzymatic reactions. Organisms which are not able to synthesize PLP de novo acquire B6 vitamers from nutrients and interconvert them through a salvage pathway, which involves pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PDXK). PNPOx converts pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to PLP, using flavinmononucleotide (FMN) as coenzyme. Both Escherichia coli and human PNPOx are homodimers and, although these enzymes share only 39% of sequence identity, have very similar structural and functional properties. PNPOx plays a crucial role in the regulation of PLP metabolism. It has been proposed that PLP inhibits the catalytic activity of both E. coli and human PNPOx by binding at the active site and acting as a competitive inhibitor. However, PLP can also bind tightly at a secondary site. Our kinetics characterisation suggests that PLP inhibition results from binding of this vitamer at an allosteric site, in both E. coli and human enzymes. This interpretation was confirmed by the analysis of mutated forms of E. coli PNPOx, in which PLP binding at the active site is impaired. Crystallographic studies carried out by other authors on the E. coli PNPOx indicated a possible location of the secondary PLP binding site in two surface pockets of the protein, but site-directed mutants of amino acid residues putatively critical for this interaction showed that this hypothesis is wrong. Molecular docking analyses identified a possible alternative PLP binding site, which is a cleft on the protein surface mainly delimited by arginine residues and located near the subunit interface. Characterisation of mutant forms of this site and crystallographic studies suggested that this might be the allosteric PLP binding site.
Concerning human PNPOx, it is known that missense mutations in the gene encoding this enzyme lead to the onset of a rare neurological disease, the neonatal epileptic encephalopathy (NEE); however, the molecular reason of most PNPOx mutations remains to be established. We expressed PNPOx mutants as recombinant proteins in E. coli, purified and characterised them with respect to structural and functional properties, in order to better understand the molecular basis of the disease.
The other key enzyme involved in the salvage pathway is PDXK, which converts pyridoxal (PL), pyridoxamine (PM) and pyridoxine (PN) into PLP, PMP and PNP, respectively. In Drosophila, mutations in the dPdxk gene encoding PDXK cause chromosome aberrations (CABs) and increase glucose content in larval haemolymph. This observation suggests that PDXK mutations in humans may be involved in diseases such as cancer and diabetes. We analysed the effect of the expression of four PDXK human variants in Drosophila dPdxk mutants: three of them (D87H, V128I and H246Q) are listed in databases, and one (A243G) was found in a genetic screening of patients with gestational diabetes. None of the variants was able to completely rescue CABs and glucose content. Our biochemical analysis revealed reduced catalytic activity and different affinity of these variants for PLP precursors. Overall, our findings suggest that, when PLP levels are reduced by the presence of these PDXK variants, cancer and diabetes risk may be increased
1H-NMR relaxometric analysis of drug binding effects to global unfolding of human serum albumin.
No full textBressanone/Brixen (BZ), 18-20 Settembre 200
Thrombin inhibition by the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester
No full textThrombin is the last enzyme in the blood coagulation cascade. All pharmacological aspects support the use of thrombin inhibitors as antithrombotic agents. Here, we review the unusual inhibition behavior of the highly selective,reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysinep-nitrophenyl ester (EOC-D-Phe-Pro-azaLys-ONp) targeted to the active center of human alpha-thrombin. Eoc-D-Phe-Pro-azaLys-ONp is an acylating agent, but its hydrolysis product 1(N-ethoxycarbonyl-D-phenylatanyl-L-prolyl)-2(4-aminobutyl)hydrazine behaves as a highly selective human alpha-thrombin competitive inhibitor
Allostery in a monomeric protein: the case of human serum albumin.
No full textHuman serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, both functional and structural aspects of the allosteric modulation of heme and drug (e.g., warfarin and ibuprofen) binding to HSA and of the drug-dependent reactivity of HSA-heme are reviewed
Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin.
No full textMost of the esterase properties of human serum albumin (HSA) are the result of multiple irreversible chemical modifications rather than turnover. The HSA-catalyzed hydrolysis of 4-nitrophenyl myristate (NphOMy) is consistent with the minimum three-step mechanism involving the acyl-enzyme intermediate HSA-OMy: Under all the experimental conditions, values of K(s) (= k(-1)/k(+1)), k(+2), and k(+2)/K(s) determined under conditions where [HSA]⩾5×[NphOMy] and [NphOMy]⩾5×[HSA] match very well each other. The deacylation process is rate limiting in catalysis (i.e., k(+3)≪k(+2)) and k(-2)∼k(-3)∼0s(-1). The pH dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a)-shift of one ionizing group from 8.9±0.2 in NphOMy-free HSA to 6.8±0.3 in the HSA:NphOMy adduct. The HSA-catalyzed hydrolysis of NphOMy is inhibited competitively by diazepam, indicating that Tyr411 is the active-site nucleophile
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Mn(II) binding to human serum albumin: A (1)H-NMR relaxometric study.
No full textHuman serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water (1)H-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the (1)H-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water (1)H-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes
- …
