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Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free...
High concentrations of glucose induce cardiomyocyte apoptosis, and contribute to diabetic cardiomyopathy. Relaxin-2 and relaxin-3 are two members of the relaxin peptide family that are cardioprotective. However, it remains unknown whether relaxin-2 or relaxin-3 can regulate apoptosis in high glucose treated-neonatal rat ventricular myocytes (NRVMs). In cultured NRVMs, 33 mmol/l high glucose (HG) increased...
Replication is a process which provides two copies of genetic material to a mother cell that are essential for passing complete genetic information to daughter cells. Despite the extremely precise control of this process, regulation of replication can be impaired. This may trigger e.g. re-replication which leads to an increase in the total DNA content in a cell and, depending on the intensity, may...
Effect of trimethylamine N-oxide (TMAO), well-known osmolyte, widely used to imitate crowded intracellular conditions, on the quaternary structure of recombinant human small heat shock protein HspB6 (Hsp20) was analyzed by means of size-exclusion chromatography, chemical crosslinking and analytical ultracentrifugation. Consistent with previous reports, in the absence of TMAO unphosphorylated, pseudophosphorylated...
MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca 2+ and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca 2+ ...
The Nuclear Factor 90 (NF90) and its isoforms constitute a family of proteins that can interact with double-stranded (ds) RNA, through its dsRNA binding motifs. Due to various potential translational events such as alternative splicing, the human Interleukin enhancer binding factor 3 (ilf3) gene codes for multifunctional proteins that are NF90 and its isoforms, involved in transcription, translation,...
Hypoxia-inducible factor-1alpha (HIF-1α) is a regulated subunit of the hypoxia-inducible factor 1 (HIF1), which functions as a key transcription factor in response to hypoxic stress by regulating genes involved in maintaining oxygen homeostasis. In recent years, a growing body of studies showed that HIF-1α was significantly increased in hepatic fibrotic tissues and activated hepatic stellate cells...
In recent years the chaperone HSC-70 has become a target for drug design with a strong focus in anticancer therapies. In our study of possible inhibitors of HSC-70 enzymatic activity we screened compounds by NMR as well as X-ray crystallography. As part of our screening efforts we crystallized the human HSC-70 ATP binding domain and obtained novel crystal forms in addition to known structures. The...
α-Glucosidases are ubiquitous enzymes that hydrolyze the α-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an α-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant BspAG31A, produced in Escherichia coli, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This...
Clinical evidence indicates that insulin therapy improves implant survival rates in diabetic patients; however, the mechanisms responsible for this effect are unknown. Here, we test if insulin exerts anti-oxidative effects, thereby improving diabetes-associated impaired osteoblast behavior on titanium implants. To test this hypothesis, we cultured primary rabbit osteoblasts in the presence of titanium...
Transducin (T) is a heterotrimer of Tα, Tβ, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N 3 GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N 3 GTP per mol of T. Although Tα was preferentially labeled by 8-N 3 GTP, Tβ and Tγ were also modified. Photolabeling...
Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in...
Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated...
To substantiate the functionality of a crystallographically evidenced allosteric site in Bacillus caldovelox arginase (Bewley et al., 1999), we have examined the kinetic consequences of the single mutations of Asp199 and Glu256, which interact with l-arginine in this site. The introduced mutations (Asp199 → Asn, Asp199 → Ala, Glu256 → Gln and Glu256 → Ala) had no effect on the hexameric structure...
Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase...
Alpha synuclein is a 14 kDa intrinsically disordered, presynaptic protein whose fibrillation is a critical step in the pathogenesis of Parkinson's disease (PD). A structural investigation of the effect of escitalopram (a selective serotonin reuptake inhibitor) on α-synuclein was performed using ANS and ThT assays, CD, turbidity and Rayleigh scattering measurements as well as atomic force and transmission...
Alcohol dehydrogenase (ADH, EC 1.1.1.1) plays important roles in the metabolism of alcohols and aldehydes. They are often subjected to conformational changes that are critical for the enzymatic activity and have received intensive investigation for horse liver ADH. However, for the large plant ADH members, little is known regarding both the conformational change and its relationship to catalytic activity...
A well known rule of high thermal stability of GC-rich DNA helices can be reversed with the use of certain ions, rendering AT-rich duplexes more stable. We have sought to elucidate the structural basis of this phenomenon for choline dihydrogen phosphate, an ionic liquid known for extension of long-term chemical stability of biomolecules. NMR experiments complemented with CD spectroscopy revealed subtle...
Two secretory phospholipase A 2 (sPLA 2 s) from Glycine max, GmsPLA 2 -IXA-1 and GmsPLA 2 -XIB-2, have been purified as recombinant proteins and the activity was evaluated in order to obtain the optimum conditions for catalysis using mixed micelles and lipid monolayers as substrate. Both sPLA 2 s showed a maximum enzyme activity at pH 7 and a requirement of...
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