Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase.

FTO is an N6-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca2+-dependent but independent on FTO phosphorylation. We found that FTO-CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations-exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO-CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca2+ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.

Replacing centrifugation with mixing in urine analysis enriches protein pool in the urine samples.

Urine is the basic diagnostic material, easy to collect, not requiring invasive approach. During standard procedure the urine samples are centrifuged and the supernatant analysed physically, biochemically, and microscopically. The centrifugation step removes proteins including those forming aggregates especially in the state of illness and after transplantation. Here, we analysed the effect of urine centrifuging on specific protein content in urine samples obtained from cardiovascular patients (CVD) and after kidney or liver transplantation. We tested homogeneous whole urine samples, standardly centrifuge one, and the pellet after centrifuging. Protein content was examined using Western blot analysis and mass spectrometry (MS) of samples from CVD patients or the one after transplantation. The average of 21% proteins from non-centrifuged samples were found in the pellet removed after standard centrifugation. MS analysis confirmed that diagnostically important proteins were located there in. In 90% of cases whole urine samples contained more proteins than standard supernatant, among them e.g. proteins involved in immunological response like immunoglobulins and complement compounds secreted by leucocytes. Replacing centrifuging with intensive mixing of urine samples provides a method of enriching the samples with proteins removed during standard procedure, thus increasing possibility of finding new biomarkers for diseases undiagnosable with classic urine analysis.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.

The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

Evaluation of Anti-cancer Activity of Stilbene and Methoxydibenzo[b,f] oxepin Derivatives.

BACKGROUND: Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE: We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS: Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION: Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells.

BACKGROUND: The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell-cell dynamics in tumor-like spatial structures. RESULTS: Clear-cell and papillary renal cell carcinoma (cc and pRCC) cell lines were used in the study to determine the impact of hypoxia on primary drug resistance phenomenon previously observed in papillary, but not in ccRCC. Resistance was confirmed in monolayer culture and in 3D models in soft agar and suspension culture. Human papillary kidney cancer stem-like cells (HKCSCs) cultured in hypoxia developed resistance to sorafenib, while when cultured in normoxia resistance to axitinib has developed. Flow cytometry revealed that hypoxia decreased proliferation rates in all investigated RCC cells. In HKCSCs, there was an increase of quiescent cells (Ki67-) and percentage of cells arrested in S phase. It also appeared that map2k1 and eif4b protein expression is altered in papillary RCC resistant to tested drugs at different oxygen tensions. Also, HKCSCs did not express vegfr-1, braf nor c-kit, TKIs target receptors, which were present in ccRCC cells sensitive to TKI treatment. CONCLUSIONS: The results confirm that low oxygen tension affects RCC cells. Hypoxia facilitates induction of sorafenib resistance in pRCC and induces map2k1 overexpression, while normoxic axitinib-resistant cells up-regulated eif4b. Further studies may determine if map2k1 or eif4b proteins play a role in pRCC resistance to TKIs. It is also of interest to establish if other than vegfr-1, braf, c-kit receptors can serve as potential molecular targets for more effective anti-RCC strategies.

Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation.

Adenoviral dodecahedron (Dd) is a virus-like particle composed of twelve pentameric penton base (Pb) proteins, responsible for adenovirus cell penetration. It is generated spontaneously in the baculovirus system upon expression of the Pb gene of adenovirus serotype 3. This particle shows remarkable cell penetration ability with 2,00,000-3,00,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. We have used it in the past for delivery of small drugs as well as a vaccination platform, in which Dd serves as a particulate vaccine delivery system. Since development of new biomedicals depends strongly on the cost of their expression and purification, we attempted, albeit unsuccessfully, to obtain Dd expression in bacteria. We therefore retained its expression in the baculovirus/insect cells system but introduced significant improvements in the protocols for Dd expression and purification, leading to considerable savings in time and improved yield.

Intrauterine growth retarded piglet as a model for humans--studies on the perinatal development of the gut structure and function.

The overall acceptance of pig models for human biomedical studies is steadily growing. Results of rodent studies are usually confirmed in pigs before extrapolating them to humans. This applies particularly to gastrointestinal and metabolism research due to similarities between pig and human physiology. In this context, intrauterine growth retarded (IUGR) pig neonate can be regarded as a good model for the better understanding of the IUGR syndrome in humans. In pigs, the induction of IUGR syndrome may include maternal diet intervention, dexamethasone treatment or temporary reduction of blood supply. However, in pigs, like in humans, circa 8% of neonates develop IUGR syndrome spontaneously. Studies on the pig model have shown changes in gut structure, namely a reduced thickness of mucosa and muscle layers, and delayed kinetic of disappearance of vacuolated enterocytes were found in IUGR individuals in comparison with healthy ones. Functional changes include reduced dynamic of gut mucosa rebuilding, decreased activities of main brush border enzymes, and changes in the expression of proteins important for carbohydrate, amino acids, lipid, mineral and vitamin metabolism. Moreover, profiles of intestinal hormones are different in IUGR and non-IUGR piglets. It is suggested that supplementation of the mothers during the gestation and/or the IUGR offspring after birth can help in restoring the development of the gastrointestinal tract. The pig provides presumably the optimal animal model for humans to study gastrointestinal tract structure and function development in IUGR syndrome.

Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.

Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor.

The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a ß-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.

Novel AlkB dioxygenases--alternative models for in silico and in vivo studies.

BACKGROUND: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1-8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. METHODOLOGY AND FINDINGS: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB(-) mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. CONCLUSIONS: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

Systematic bioinformatics and experimental validation of yeast complexes reduces the rate of attrition during structural investigations.

For high-throughput structural studies of protein complexes of composition inferred from proteomics data, it is crucial that candidate complexes are selected accurately. Herein, we exemplify a procedure that combines a bioinformatics tool for complex selection with in vivo validation, to deliver structural results in a medium-throughout manner. We have selected a set of 20 yeast complexes, which were predicted to be feasible by either an automated bioinformatics algorithm, by manual inspection of primary data, or by literature searches. These complexes were validated with two straightforward and efficient biochemical assays, and heterologous expression technologies of complex components were then used to produce the complexes to assess their feasibility experimentally. Approximately one-half of the selected complexes were useful for structural studies, and we detail one particular success story. Our results underscore the importance of accurate target selection and validation in avoiding transient, unstable, or simply nonexistent complexes from the outset.

Overexpression of HAP4 in glucose-derepressed yeast cells reveals respiratory control of glucose-regulated genes.

A link between control of respiration and glucose repression in yeast is reported. The HAP4 gene was overexpressed in a Delta mig1 deletion background, generating a mutant in which respiratory function is stimulated and glucose repression is diminished. Although this combination does not result in derepression of genes encoding proteins involved in respiratory function, it nevertheless generates resistance against 2-deoxyglucose and hence contributes to more derepressed growth characteristics. Unexpectedly, overexpression of HAP4 in the Delta mig1 deletion strain causes strong repression of several target genes of the Mig1p repressor. Repression is not restricted to glucose growth conditions and does not require the glucose repressors Mig2p or Hxk2p. It was observed that expression of the SUC2 gene is transiently repressed after glucose is added to respiratory-growing Delta mig1 cells. Additional overexpression of HAP4 prevents release from this novel repressed state. The data presented show that respiratory function controls transcription of genes required for the metabolism of alternative sugars. This respiratory feedback control is suggested to regulate the feed into glycolysis in derepressed conditions.

RNA degradation in yeast and human mitochondria.

RNA turnover in yeast mitochondria is controlled by the complex called degradosome, which consists of two nuclear-encoded proteins: the SUV3 gene codes for an RNA helicase and the DSS1 gene codes for an RNase. In contrast to yeast, much less is known about RNA degradation in human mitochondria. We suggest that the key enzyme involved in this process is nuclear-encoded polynucleotide phosphorylase, hPNPase.

Human polynucleotide phosphorylase, hPNPase, is localized in mitochondria.

The human gene encoding a polynucleotide phosphorylase (hPNPase) has been recently identified as strongly up-regulated in two processes leading to irreversible arrest of cell division: progeroid senescence and terminal differentiation. Here, we demonstrate that the hPNPase is localized in mitochondria. Our finding suggests the involvement of mitochondrial RNA metabolism in cellular senescence.

The yeast mitochondrial degradosome. Its composition, interplay between RNA helicase and RNase activities and the role in mitochondrial RNA metabolism.

The yeast mitochondrial degradosome (mtEXO) is an NTP-dependent exoribonuclease involved in mitochondrial RNA metabolism. Previous purifications suggested that it was composed of three subunits. Our results suggest that the degradosome is composed of only two large subunits: an RNase and a RNA helicase encoded by nuclear genes DSS1 and SUV3, respectively, and that it co-purifies with mitochondrial ribosomes. We have found that the purified degradosome has RNA helicase activity that precedes and is essential for exoribonuclease activity of this complex. The degradosome RNase activity is necessary for mitochondrial biogenesis but in vitro the degradosome without RNase activity is still able to unwind RNA. In yeast strains lacking degradosome components there is a strong accumulation of mitochondrial mRNA and rRNA precursors not processed at 3'- and 5'-ends. The observed accumulation of precursors is probably the result of lack of degradation rather than direct inhibition of processing. We suggest that the degradosome is a central part of a mitochondrial RNA surveillance system responsible for degradation of aberrant and unprocessed RNAs.

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA.

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub-fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a K(m) of 41.9 micro M and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution.