Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Biochem Biophys Res Commun ; 596: 43-48, 2022 03 12.
Article in English | MEDLINE | ID: mdl-35108653

ABSTRACT

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Bacterial Proteins/metabolism , Peptide Synthases/metabolism , Polylysine/metabolism , Streptomyces/enzymology , Adenosine Monophosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites/genetics , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Kinetics , Models, Molecular , Peptide Synthases/chemistry , Peptide Synthases/genetics , Protein Binding , Protein Domains , Streptomyces/genetics , Substrate Specificity
2.
Nucl Med Biol ; 94-95: 92-97, 2021.
Article in English | MEDLINE | ID: mdl-33609918

ABSTRACT

INTRODUCTION: We clarified the renal uptake and urinary secretion mechanism of [99mTc]dimercaptosuccinic acid ([99mTc]DMSA) via drug transporters in renal proximal tubules. METHODS: [99mTc]DMSA was added to human embryonic kidney 293 cells expressing human multidrug and toxin extrusion (MATE)1 and MATE2-K, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)2; to Flp293 cells expressing human organic anion transporter (OAT)1 and OAT3; and to vesicles expressing P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)2, MRP4, or breast cancer resistance protein with and without probenecid (OAT inhibitor for both OATs and MRPs). Time activity curves of [99mTc]DMSA with and without probenecid were established using LLC-PK1 cells. Biodistribution and single photon emission computed tomography (SPECT) imaging in mice were conducted using [99mTc]DMSA with and without probenecid. RESULTS: [99mTc]DMSA uptake was significantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 was inhibited by probenecid. [99mTc]DMSA uptake into vesicles that highly expressed MRP2 was significantly higher in adenosine triphosphate (ATP) than in adenosine monophosphate (AMP), and probenecid decreased uptake to similar levels as that in AMP. In the time activity curves for [99mTc]DMSA in LLC-PK1 cells, probenecid loading inhibited accumulation from the basolateral side into LLC-PK1 cells, whereas accumulation from the apical side into cells gradually increased. Transport of [99mTc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [99mTc]DMSA with probenecid loading resulted in significantly higher accumulation in blood, heart, liver, and bladder after [99mTc]DMSA injection compared with control mice. Probenecid induced significantly lower accumulation in the kidney after [99mTc]DMSA injection. CONCLUSIONS: [99mTc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on the basolateral side, and then a small volume of [99mTc]DMSA will be excreted in urine via MRP2. ADVANCES IN KNOWLEDGE: [99mTc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and is slightly excreted from the cells via MRP2. IMPLICATIONS FOR PATIENT CARE: [99mTc]DMSA may be useful for measuring renal transport function with OAT3 in patients.


Subject(s)
Organic Anion Transporters, Sodium-Independent/metabolism , Technetium Tc 99m Dimercaptosuccinic Acid/metabolism , Technetium Tc 99m Dimercaptosuccinic Acid/urine , Biological Transport , Cell Line , Multidrug Resistance-Associated Protein 2 , Technetium Tc 99m Dimercaptosuccinic Acid/pharmacokinetics , Tissue Distribution
SELECTION OF CITATIONS
SEARCH DETAIL
...