A Key Silencing Histone Mark on Chromatin Is Lost When Colorectal Adenocarcinoma Cells Are Depleted of Methionine by Methionine γ-Lyase

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis

Immobilization of proteins in silica gel: Biochemical and biophysical properties

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques

Role of the Subunits Interactions in the Conformational Transitions in Adult Human Hemoglobin: an Explicit Solvent Molecular Dynamics Study

Hemoglobin exhibits allosteric structural changes upon ligand binding due to the dynamic interactions between the ligand binding sites, the amino acids residues and some other solutes present under physiological conditions. In the present study, the dynamical and quaternary structural changes occurring in two unligated (deoxy-) T structures, and two fully ligated (oxy-) R, R2 structures of adult human hemoglobin were investigated with molecular dynamics. It is shown that, in the sub-microsecond time scale, there is no marked difference in the global dynamics of the amino acids residues in both the oxy- and the deoxy- forms of the individual structures. In addition, the R, R2 are relatively stable and do not present quaternary conformational changes within the time scale of our simulations while the T structure is dynamically more flexible and exhibited the T\rightarrow R quaternary conformational transition, which is propagated by the relative rotation of the residues at the {\alpha}1{\beta}2 and {\alpha}2{\beta}1 interface.Comment: Reprinted (adapted) with permission from J. Phys. Chem. B DOI:10.1021/jp3022908. Copyright (2012) American Chemical Societ

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: differential oxidative stress in a Guinea pig transfusion model

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto-transfusion control group. Particularly, tissue 4-hydroxynonenal-adducts, tissue malondialdehyde adducts and plasma 8-oxo-2'-deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with PEG-Hboxy, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high-affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced

Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with &gt;80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with &gt; 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1&nbsp;mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained &gt; 90% of its conjugation, whereas for maleimide-PEG &lt; 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate

Rational design of a user-friendly aptamer/peptide-based device for the detection of staphylococcus aureus

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products

MediaChrom: exploring a new family of pyrimidoindolone-based polarity-sensitive dyes

The modern biological research asks for a continuous development of new fluorescent dyes characterized by improved performances and suitable to be used as markers or probes [1]. A particular class of dyes, called polarity-sensitive dyes have the unique features to display a different emission maximum as a function of the polarity of their molecular environment (media). This peculiarity makes polarity-sensitive dyes the ideal probes to monitor the local properties of particular cell districts as well as different type of biomolecular interactions [2]. Since many years, we have been interested in the development of new strategies for the synthesis and the functionalization of indoles and polycyclic indole-based heterocycles. In this context, we reported a domino approach to pyrimidoindolones [3] that displayed interesting fluorescence properties. Starting from these findings, a small library of original polarity-sensitive fluorescent dyes, nicknamed MediaChrom, has been prepared [4]. They are characterized by a pyrimidoindolone core fitted out with a conjugated push-pull system, and a linker for an easy coupling with biomolecules. The synthetic strategy involves a highly chemo- and regioselective gold catalyzed cycloisomerization as key step. The photophysical properties of MediaChrom dyes have been evaluated, and some potential biological applications have been spottily investigated. [1] Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. [2] Klymchenko, A. S.; Mely, Y. In: Progress in Molecular Biology and Translational Science, Morris, M.C. Editor(s), Academic Press, 2013, Vol. 113, Cap. 2, 35. [3] Facoetti, D.; Abbiati, G.; d\u2019Avolio, L.; Ackermann, L.; Rossi, E. Synlett 2009, 2273. [4] Dell'Acqua, M.; Ronda, L.; Piano, R.; Pellegrino, S.; Clerici, F.; Rossi, E.; Mozzarelli, A.; Gelmi, M. L.; Abbiati, G. J. Org. Chem. 2015, 21, 10939. This work has been supported by Fondazione Cariplo, Grant No. 2012-0907