Magdalena Tworzydło

Gel‐derived materials of a CaO‐P2O5‐SiO2 system modified by boron, sodium, magnesium, aluminum, and fluorine compounds

Bioactive glass-ceramic materials of the CaO-P(2)O(5)-SiO(2) system modified by adding boron, magnesium, sodium, fluorine, and aluminum were obtained using the sol-gel method. Gel-derived materials were produced in the pellet form obtained by compression of powders as well as in coatings on glass slides. The materials obtained were examined in vitro with regard to the ability of calcium phosphate layer to form on the material surface as the result of contact with simulated body fluid (SBF). SBF pH changes and calcium solubility in this solution were determined and scanning electron microscopy, energy-dispersive X-ray analysis, and infrared spectroscopy studies were conducted before and after contact of the materials with SBF. The gels modified by aluminum were amorphous, whereas the sodium and fluorine additives promoted the bulk crystallization of gel-derived materials. The ability of calcium phosphates to crystallize on the surface of gel-derived materials depended only slightly on the types of additives applied, and the character of this dependence was different from that observed in melted glasses. Moreover, to estimate the biocompatibility of gel-derived coatings, we examined the proliferation, collagen synthesis, adhesion, and morphology of fibroblasts (NRK cells) cultured in the presence of gel-derived materials. The results of these experiments showed that none of the tested materials significantly reduced any cell function.

cAMP Receptor Protein from Escherichia coli as a Model of Signal Transduction in Proteins – A Review

In Escherichia coli, cyclic AMP receptor protein (CRP) is known to regulate the transcription of about 100 genes. The signal to activate CRP is the binding of cyclic AMP. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP-binding domain to the C-terminal domain of the protein, which is responsible for interaction with specific sequences of DNA. The signal transduction plays a crucial role in the activation of the protein. The most sophisticated spectroscopic techniques, other techniques frequently used in structural biochemistry, and site-directed mutagenesis have been used to investigate the details of cAMP-mediated allosteric control over CRP conformation and activity as a transcription factor. The aim of this review is to summarize recent works and developments pertaining to cAMP-dependent CRP signal transduction in E. coli.

Role of silent polymorphisms within the dopamine D1 receptor associated with schizophrenia on D1-D2 receptor hetero-dimerization.

Within the coding region of the dopamine D(1) receptor (D(1)R), two synonymous polymorphisms, D(1)R(G198A) and D(1)R(G1263), have been identified and postulated to correlate with the schizophrenia phenotype. Binding studies revealed that the density of these genetic variants was much lower than the density of wild type D(1)R in the human embryonic kidney (HEK) 293 cell line, used as a model system. From the data obtained using MFOLD software it is apparent that the G198A mutation has a greater impact on the secondary structure of the mRNA, which may affect its translation. However, the G1263A mutation is localized within the serine 421 codon of D(1)R, which is predicted to be a potential site of phosphorylation according to the PHOSIDA database. In order to determine whether the studied synonymous polymorphisms influence the process of dopamine D(1)-D(2) receptors heterodimerization, we employed fluorescence resonance energy transfer (FRET) technology. The dopamine D(2) receptor (D(2)R) was tagged with cyan fluorescence protein and the D(1)R and its genetic variants were tagged with yellow fluorescence protein. The degree of D(1)-D(2) receptor hetero-dimerization was significantly decreased when genetic variants of D(1)R were co-expressed with D(2)R. Since the D(1)R mutations affected the expression levels of the proteins in the cell membrane without affecting the cellular localization of the receptor proteins, we postulated that the D(1)R polymorphisms altered the translation rate and protein structure of the receptor. The altered hetero-dimerization that likely results from the lower expression of these genetic variants of D(1)R with D(2)R may be partially responsible for the association of both G198A and G1263A polymorphisms with the schizophrenia phenotype.

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long‐distance signal transmission from the N‐terminal cAMP‐binding domain to the C‐terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N‐terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long‐distance conformational signal transduction within the protein from the C‐terminal DNA‐binding domain to the N‐terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped‐flow and Förster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA–CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.

New insights into the model of dopamine D1 receptor and G-proteins interactions.

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9-10 nm.

Engineered β-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation

Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, Ka, ΔH, TΔS, ΔG), chemical and thermal stability (ΔGD, Cmid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein-lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi3 prefer different types of membrane domains they are both co-localized with the D1 receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi3 and their heterotrimers, as well as the D1 receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-β-cyclodextrin or Fumonisin B1 led to the disruption of cholesterol-sphingolipids containing domains and changed the diffusion of Gαi3 and the D1 receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi3 prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D1 receptor exists in several different membrane localizations, depending on the receptors conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.

PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X‐ray repair cross‐complementing protein 1 (XRCC1), a key factor in single‐strand break and base excision repair, is recruited into nuclear bodies formed in response to replication‐related single‐strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP‐ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and ‐2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1‐contaning foci constitute newly recognized PML‐like nuclear bodies that accrete and locally deliver essential factors for repair of single‐strand DNA breaks in replication regions.—Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann‐Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML‐like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication‐based single‐strand breaks. FASEB J. 33, 2301–2313 (2019). www.fasebj.org