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1.
Sci Rep ; 13(1): 6017, 2023 04 12.
Article in English | MEDLINE | ID: mdl-37045950

ABSTRACT

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6-7 months (T1) and 12-13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.


Subject(s)
Diabetes Mellitus , Diabetic Nephropathies , Animals , Swine , Male , Humans , Diabetic Nephropathies/pathology , Swine, Miniature , Kidney/pathology , Obesity/pathology , Glomerular Basement Membrane/pathology , Diabetes Mellitus/pathology
2.
ACS Comb Sci ; 19(10): 657-669, 2017 10 09.
Article in English | MEDLINE | ID: mdl-28746804

ABSTRACT

We herein present broadly useful, readily available and nonintegral hydroxylamine linkers for the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying on high control and flexibility with respect to reagents and synthetic processes. A trityl-based hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a chemiluminescence-based assay.


Subject(s)
Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylases/chemistry , Hydroxamic Acids/chemical synthesis , Peptides/chemical synthesis , Humans , Peptide Library , Solid-Phase Synthesis Techniques , Structure-Activity Relationship
4.
PLoS One ; 9(6): e98800, 2014.
Article in English | MEDLINE | ID: mdl-24915177

ABSTRACT

Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening conditions. Furthermore a marked improvement in the correlation was found by using kinase constructs containing the catalytic domain in presence of additional domains or subunits.


Subject(s)
Drug Evaluation, Preclinical/methods , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Enzyme Activation/drug effects , Inhibitory Concentration 50 , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Small Molecule Libraries
5.
Anal Biochem ; 452: 34-42, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24534253

ABSTRACT

ADAM12 belongs to the A disintegrin and metalloprotease (ADAM) family of secreted sheddases activating extracellular growth factors such as epidermal growth factor receptor (EGFR) ligands and tumor necrosis factor-alpha (TNF-α). ADAM proteases, most notably ADAM17 (TNF-α-converting enzyme), have long been investigated as pharmaceutical drug targets; however, due to lack of potency and in vivo side effects, none of the small-molecule inhibitors discovered so far has made it beyond clinical testing. Ongoing research on novel selective inhibitors of ADAMs requires reliable biochemical assays to validate molecular probes from large-scale screening efforts. Here we describe an electrophoretic mobility shift assay for ADAM12 based on the identification of an optimized peptide substrate that is characterized by excellent performance and reproducibility.


Subject(s)
ADAM Proteins/metabolism , Electrophoretic Mobility Shift Assay/methods , Membrane Proteins/metabolism , Peptides/metabolism , ADAM12 Protein , Amino Acid Sequence , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Hydroxamic Acids/pharmacology , Molecular Sequence Data , Peptides/chemistry , Protease Inhibitors/pharmacology , Reproducibility of Results
6.
Cell Rep ; 2(2): 419-31, 2012 Aug 30.
Article in English | MEDLINE | ID: mdl-22902405

ABSTRACT

Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.


Subject(s)
Lysine/metabolism , Protein Processing, Post-Translational/physiology , Proteome/metabolism , Acetylation , Amino Acid Motifs , Animals , Humans , Organ Specificity/physiology , Proteomics/methods , Rats , Rats, Sprague-Dawley
7.
FEBS J ; 279(10): 1777-87, 2012 May.
Article in English | MEDLINE | ID: mdl-22385435

ABSTRACT

The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy.


Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/enzymology , Mutation , Thymidine Kinase/chemistry , Thymidine Kinase/genetics , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Catalytic Domain , Humans , Kinetics , Substrate Specificity , Zidovudine/chemistry , Zidovudine/metabolism
8.
FEBS J ; 272(14): 3733-42, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16008571

ABSTRACT

The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3'-modified nucleoside analogs like 3'-azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3'-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3'-substituents like the azido group in AZT but influences negatively the interactions between Dm-dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure-function relationship provides an improved background for future development of novel mutant suicide genes for Dm-dNK-mediated gene therapy.


Subject(s)
Asparagine/genetics , Aspartic Acid/genetics , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Mutation/genetics , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Asparagine/metabolism , Aspartic Acid/metabolism , Crystallography, X-Ray , Kinetics , Models, Molecular , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Structure, Tertiary , Substrate Specificity , Thymine Nucleotides/pharmacology
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