A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.
A 24-amino acid leader peptide of a new human recombinant manganese superoxide dismutase can enter cells and carry molecules. Here, we demonstrated that six of the 24 amino acids penetrate cells through a particular gate represented by a specific amino acid sequence of the oestrogen receptor (ER). We analysed the internalization of the synthetic hexapeptide and the cytotoxic activity of the hexapeptide conjugated to cisplatin on a cell line panel. In most cell lines, the hexapeptide delivered an amount of cisplatin that was 2 to 8 times greater than that released by cisplatin when the drug was used alone. This increased delivery increases the therapeutic index of cisplatin and reduces side effects caused by a high dosage or long-term treatment times. We may consider this hexapeptide a new molecular carrier to deliver molecules with therapeutic activity into ER(+) cells for diagnostic purposes and clinical or immune therapy.
A deglycosylation step using Peptide-N-Glycosidase F (PNGaseF) has been introduced in a standard proteomic protocol to more confidently identify egg based binders. The ingenuity of introducing a PNGaseF digestion was aimed at removing the molecular hindrance, made up by the heavily glycosylated egg proteins, before the protease(s) hydrolysis. This novelty in the protocol resulted in obtaining a significant increase of proteolytic egg peptides thus improving the quality and reliability of egg identification in artwork samples. The protocol has been set up on paint replicas and successfully tested on two historical samples of different origin.
Cellulases and xylanases are the key enzymes involved in the conversion of lignocelluloses into fermentable sugars. Western Ghat region (India) has been recognized as an active hot spot for the isolation of new microorganisms. The aim of this work was to isolate new microorganisms producing cellulases and xylanases to be applied in brewer's spent grain saccharification.
Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the 12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.
A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.
Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.
An α-L-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-L-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L(-1)) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-L-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-L-arabinofuranoside it follows Michaelis-Menten kinetics with a K (M) of 0.64 mM and a k (cat) of 3,010 min(-1). The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-L-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.
Proteomic strategies are herein proved to be a complementary approach to the well established amino acid composition analysis for the characterization of the aging and deterioration phenomena occurring to proteinaceous materials in works-of-art. Amino acid analyses on several samples demonstrated that proteins in the frescoes from the Camposanto Monumentale in Pisa are deteriorated as revealed by the decrease in Met, Lys, and Tyr content and by the presence in all the samples of amino malonic acid as a result of Ser, Phe, and Cys oxidation. Proteomic analysis identified deamidation at Asn and Gln as a further major event occurred. This work paves the way to the exploitation of proteomic strategies for the investigation of the molecular effects of aging and deterioration in historical objects. Results show that proteomic searches for deamidation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) could constitute a routine analysis for paintings or any artistic and historic objects where proteins are present. Peptides that can be used as molecular markers when casein is present were identified.
IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.