Proteomic Characterization of Collagen-Based Animal Glues for Restoration.

Animal glues are widely used in restoration as adhesives, binders, and consolidants for organic and inorganic materials. Their variable performances are intrinsically linked to the adhesive properties of collagen, which determine the chemical, physical, and mechanical properties of the glue. We have molecularly characterized the protein components of a range of homemade and commercial glues using mass spectrometry techniques. A shotgun proteomic analysis provided animal origin, even when blended, and allowed us to distinguish between hide and bone glue on the basis of the presence of collagen type III, which is abundant in connective skin/leather tissues and poorly synthetized in bones. Furthermore, chemical modifications, a consequence of the preparation protocols from the original animal tissue, were thoroughly evaluated. Deamidation, methionine oxidation, and backbone cleavage have been analyzed as major collagen modifications, demonstrating their variability among different glues and showing that, on average, bone glues are less deamidated than hide glues, but more fragmented, and mixed-collagen glues are overall less deamidated than pure glues. We believe that these data may be of general analytical interest in the characterization of collagen-based materials and may help restorers in the selection of the most appropriate materials to be used in conservation treatments.

Cerato-Platanins from Marine Fungi as Effective Protein Biosurfactants and Bioemulsifiers.

Two fungal strains, Aspergillus terreus MUT 271 and Trichoderma harzianum MUT 290, isolated from a Mediterranean marine site chronically pervaded by oil spills, can use crude oil as sole carbon source. Herein, these strains were investigated as producers of biosurfactants, apt to solubilize organic molecules as a preliminary step to metabolize them. Both fungi secreted low molecular weight proteins identified as cerato-platanins, small, conserved, hydrophobic proteins, included among the fungal surface-active proteins. Both proteins were able to stabilize emulsions, and their capacity was comparable to that of other biosurfactant proteins and to commercially available surfactants. Moreover, the cerato-platanin from T. harzianum was able to lower the surface tension value to a larger extent than the similar protein from A. terreus and other amphiphilic proteins from fungi. Both cerato-platanins were able to make hydrophilic a hydrophobic surface, such as hydrophobins, and to form a stable layer, not removable even after surface washing. To the best of our knowledge, the ability of cerato-platanins to work both as biosurfactant and bioemulsifier is herein demonstrated for the first time.

Characterization of a Surface-Active Protein Extracted from a Marine Strain of Penicillium chrysogenum.

Marine microorganisms represent a reservoir of new promising secondary metabolites. Surface-active proteins with good emulsification activity can be isolated from fungal species that inhabit the marine environment and can be promising candidates for different biotechnological applications. In this study a novel surface-active protein, named Sap-Pc, was purified from a marine strain of Penicillium chrysogenum. The effect of salt concentration and temperature on protein production was analyzed, and a purification method was set up. The purified protein, identified as Pc13g06930, was annotated as a hypothetical protein. It was able to form emulsions, which were stable for at least one month, with an emulsification index comparable to that of other known surface-active proteins. The surface tension reduction was analyzed as function of protein concentration and a critical micellar concentration of 2 µM was determined. At neutral or alkaline pH, secondary structure changes were monitored over time, concurrently with the appearance of protein precipitation. Formation of amyloid-like fibrils of SAP-Pc was demonstrated by spectroscopic and microscopic analyses. Moreover, the effect of protein concentration, a parameter affecting kinetics of fibril formation, was investigated and an on-pathway involvement of micellar aggregates during the fibril formation process was suggested.

Minimally Invasive and Portable Method for the Identification of Proteins in Ancient Paintings.

A novel method for the analysis of proteinaceous materials present on painted surfaces was developed by taking advantage of the adhesive ability of some fungal proteins which can form a stable and homogeneous layer on flexible transparency sheets able to capture trypsin in a fully active form. We demonstrated that the bioactive sheets were able to efficiently digest proteins, present as such, on surfaces of painted tests and historical samples, releasing peptides that can allow an easy and confident identification of the proteinaceous binders by standard bottom-up proteomic approach. By this method there is no need: (i) to transport the artifacts and (ii) to remove, even at micro level, a sample from the object. The ingenuity of the method lies in the easily accommodated sampling coupled with a minimal invasiveness.

Evolved Gas Analysis-Mass Spectrometry to Identify the Earliest Organic Binder in Aegean Style Wall Paintings.

An organic binder was identified in the painted fragments from the Canaanite palace of Tel Kabri, Israel. Recently dated to the late 18th century B.C.E. by 14 C, Tel Kabri is the most ancient of the Eastern Mediterranean sites in which Aegean style paintings have been found. The application of pigments was suspected to be using an organic binding medium, particularly for the Egyptian Blue pigment. Samples of blue paint were examined using evolved gas analysis-mass spectrometry (EGA-MS) in order to overcome the analytical challenges imposed by highly degraded aged proteinaceous materials. Egg was identified as the binder based on the presence of hexadecanonitrile and octadecanonitrile, confirming the use of a secco painting technique. Lysozyme C from Gallus gallus was detected by proteomics analysis, confirming the presence of egg. To our knowledge, this is the earliest use of egg as a binder in Aegean style wall paintings.

Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment.

Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.

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.

Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples.

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.

Application of a new xylanase activity from Bacillus amyloliquefaciens XR44A in brewer's spent grain saccharification.

BACKGROUND: 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. RESULTS: 93 microorganisms were isolated from Western Ghat and screened for the production of cellulase and xylanase activities. Fourteen cellulolytic and seven xylanolytic microorganisms were further screened in liquid culture. Particular attention was focused on the new isolate Bacillus amyloliquefaciens XR44A, producing xylanase activity up to 10.5 U mL-1. A novel endo-1,4-beta xylanase was identified combining zymography and proteomics and recognized as the main enzyme responsible for B. amyloliquefaciens XR44A xylanase activity. The new xylanase activity was partially characterized and its application in saccharification of brewer's spent grain, pretreated by aqueous ammonia soaking, was investigated. CONCLUSION: The culture supernatant of B. amyloliquefaciens XR44A with xylanase activity allowed a recovery of around 43% xylose during brewer's spent grain saccharification, similar to the value obtained with a commercial xylanase from Trichoderma viride, and a maximum arabinose yield of 92%, around 2-fold higher than that achieved with the commercial xylanase. © 2014 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ultraviolet laser-induced cross-linking in peptides.

RATIONALE: The aim of this study was to demonstrate, and to characterize by high-resolution mass spectrometry that it is possible to preferentially induce covalent cross-links in peptides by using high-energy femtosecond ultraviolet (UV) laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS: Three peptides, xenopsin, angiotensin I, and interleukin, individually or in combination, were exposed to high-energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS: High-resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS: High-energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources.

Resolution of the effects induced by W → F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F.

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.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.

BACKGROUND: The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. RESULTS: Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis-Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 µM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. CONCLUSIONS: In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.

A family GH51 α-L-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization.

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.

Molecular signatures written in bone proteins of 79 AD victims from Herculaneum and Pompeii.

An extensive proteomic analysis was performed on a set of 12 bones of human victims of the eruption that in AD 79 rapidly buried Pompeii and Herculaneum, allowing the detection of molecular signatures imprinted in the surviving protein components. Bone collagen survived the heat of the eruption, bearing a piece of individual biological history encoded in chemical modifications. Here we show that the human bone proteomes from Pompeii are more degraded than those from the inhabitants of Herculaneum, despite the latter were exposed to temperatures much higher than those experienced in Pompeii. The analysis of the specimens from Pompeii shows lower content of non-collagenous proteins, higher deamidation level and higher extent of collagen modification. In Pompeii, the slow decomposition of victims' soft tissues in the natural dry-wet hydrogeological soil cycles damaged their bone proteome more than what was experienced at Herculaneum by the rapid vanishing of body tissues from intense heat, under the environmental condition of a permanent waterlogged burial context. Results herein presented are the first proteomic analyses of bones exposed to eruptive conditions, but also delivered encouraging results for potential biomarkers that might also impact future development of forensic bone proteomics.

Deamidation at asparagine and glutamine as a major modification upon deterioration/aging of proteinaceous binders in mural paintings.

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.

PHK from phenol hydroxylase of Pseudomonas sp. OX1. Insight into the role of an accessory protein in bacterial multicomponent monooxygenases.

Bacterial multicomponent monooxygenases (BMMs) are members of a wide family of diiron enzymes that use molecular oxygen to hydroxylate a variety of aromatic compounds. The presence of genes encoding for accessory proteins not involved in catalysis and whose role is still elusive, is a common feature of the gene clusters of several BMMs, including phenol hydroxylases and several soluble methane monooxygenases. In this study we have expressed, purified, and partially characterized the accessory component PHK of the phenol hydroxylase from Pseudomonas sp. OX1, a bacterium able to degrade several aromatic compounds. The phenol hydroxylase (ph) gene cluster was expressed in Escherichia coli/JM109 cells in the absence and in the presence of the phk gene. The presence of the phk gene lead to an increase in the hydroxylase activity of whole recombinant cells with phenol. PHK was assessed for its ability to interact with the active hydroxylase complex. Our results show that PHK is neither involved in the catalytic activity of the phenol hydroxylase complex nor required for the assembly of apo-hydroxylase. Our results suggest instead that this component may be responsible for enhancing iron incorporation into the active site of the apo-hydroxylase.

Optimization of Inulin Hydrolysis by Penicillium lanosocoeruleum Inulinases and Efficient Conversion Into Polyhydroxyalkanoates.

Inulin, a polydisperse fructan found as a common storage polysaccharide in the roots of several plants, represents a renewable non-food biomass resource for the synthesis of bio-based products. Exploitation of inulin-containing feedstocks requires the integration of different processes, including inulinase production, saccharification of inulin, and microbial fermentation for the conversion of released sugars into added-value products. In this work paper, a new microbial source of inulinase, Penicillium lanosocoeruleum, was identified through the screening of a fungal library. Inulinase production using inulin as C-source was optimized, reaching up to 28 U mL-1 at the 4th day of growth. The fungal inulinase mixture (PlaI) was characterized for pH and temperature stability and activity profile, and its isoenzymes composition was investigated by proteomic strategies. Statistical optimization of inulin hydrolysis was performed using a central composite rotatable design (CCRD), by analyzing the effect of four factors. In the optimized conditions (T, 45.5°C; pH, 5.1; substrate concentration, 60 g L-1; enzyme loading, 50 U gsubstrate -1), up to 96% inulin is converted in fructose within 20 h. The integration of PlaI in a process for polyhydroxyalkanoate (PHA) production by Cupriavidus necator from inulin was tested in both separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). A maximum of 3.2 g L-1 of PHB accumulation, corresponding to 82% polymer content, was achieved in the SSF. The proved efficiency in inulin hydrolysis and its effective integration into a SSF process pave the way to a profitable exploitation of the PlaI enzymatic mixture in inulin-based biorefineries.

A versatile and user-friendly approach for the analysis of proteins in ancient and historical objects.

Identification and characterization of ancient proteins still require technical developments towards non-invasiveness, sensitivity, versatility and ease of use of the analyses. We report that the enzyme functionalized films, described in Cicatiello et al. (2018), can be used efficiently on the surface of different objects ranging from fixative-coated paper to canvas to the coating on an albumen photograph, as well as the much harder surfaces of ivory objects and the proteinaceous binders in the decoration of a wooden Egyptian coffin. The mixture of digested peptides that are efficiently captured on the functionalized surface are also amenable to LC-MS/MS analysis, which is necessary to confidently identify chemical modifications induced upon degradation, in order to characterize the conservation state of proteins. Moreover, in a two-step procedure, we have combined the trypsin functionalized film with a PNGaseF functionalized film, which adds a deglycosylation pretreatment allowing improved detection of glycosylated proteins. SIGNIFICANCE: User friendly trypsin functionalized films were implemented to expand their potential as versatile, modular tools that can be widely exploited in the world of diagnosis of cultural heritage objects, ancient proteins, and palaeoproteomics: a procedure that could be carried out by conservators or archaeologists first on-site and later analysed with standard MS techniques.

Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.

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.

Osteopontin binds ICOSL promoting tumor metastasis.

ICOSL/ICOS are costimulatory molecules pertaining to immune checkpoints; their binding transduces signals having anti-tumor activity. Osteopontin (OPN) is here identified as a ligand for ICOSL. OPN binds a different domain from that used by ICOS, and the binding induces a conformational change in OPN, exposing domains that are relevant for its functions. Here we show that in vitro, ICOSL triggering by OPN induces cell migration, while inhibiting anchorage-independent cell growth. The mouse 4T1 breast cancer model confirms these data. In vivo, OPN-triggering of ICOSL increases angiogenesis and tumor metastatization. The findings shed new light on ICOSL function and indicate that another partner beside ICOS may be involved; they also provide a rationale for developing alternative therapeutic approaches targeting this molecular trio.