Towards a better understanding of light-glucose induced modifications on the structure and biological activity of formulated Nivolumab.

In the last years, monoclonal antibodies (mAbs) have rapidly escalated as biopharmaceuticals into cancer treatments, mainly for their target specificity accompanied by less side effects than the traditional chemotherapy, and stimulation of reliable long-term anti-tumoral responses. They are potentially unstable macromolecules under shaking, temperature fluctuations, humidity, and indoor and outdoor light exposure, all stressors occurring throughout their production, transport, storage, handling, and administration steps. The chemical and physical modifications of mAbs can lead not only to the loss of their bioactivity, but also to the enhancement of their immunogenicity with increasing risk of severe hypersensitivity reactions in treated patients because of aggregation. The photostability of Nivolumab, the active principle of Opdivo®, has been here studied. The chemical modifications detected by LC-MS/MS after the light stressor showed Trp and Met mono and double oxidations as primary damage induced by light on this mAb. The oxidations were stronger when the mAb was diluted in sterile glucose solution where 5-HMF, a major heat glucose degradation product, acted as singlet oxygen producer under irradiation. However, no significant changes in the mAb conformation were found. On the contrary, formation of a significant extent of aggregates has been detected after shining high simulated sunlight doses. This again took place particularly when Nivolumab was diluted in sterile glucose, thus raising a direct correlation between the aggregation and the oxidative processes. Finally, the biological activity under light stress assessed by a blockade assay test demonstrated the maintenance of the PD-1 target recognition even under high light doses and in glucose solution, in line with the preservation of the secondary and tertiary structures of the mAb. Based on our results, as sterile glucose is mostly used for children's therapies, special warnings, and precautions for healthcare professionals should be included for their use to the pediatric population.

Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

Managing antibody stability: Effects of stressors on Ipilimumab from the commercial formulation to diluted solutions.

The stability of the monoclonal antibody Ipilimumab, the active ingredient of Yervoy®, used for the treatment of different types of cancer, has been investigated. Shaking/temperature, light exposure and dilution, protein drug renowned stressors, were applied on a 30-45-day series of experiments to observe the physicochemical and biological behavior of the molecule. Ipilimumab demonstrated stability under shaking and heat up to 45 days, without any unfolding during the induced combined stressors. Under artificial sunlight, the mAb showed to be sensitive even under the minimum dose tested (720 kJ/m2) with formation of aggregates, particularly when diluted in glucose solution. The light-induced soluble aggregates were higher in the case of diluted samples irradiated with much higher light doses (10460 kJ/m2). The aggregation of Ipilimumab took place also by irradiating the non-diluted formulation, indicating that the excipients did not protect completely the drug from photodegradation. Amino acid oxidation and deamidation were found. Anyway, after irradiation with both light doses, soluble Ipilimumab maintained its typical ß-sheets structure, and the tertiary structure was nearly maintained compared to the dark. As an additional stressor test, the effect of dilution on the formulation was monitored by using a saline solution (1 mg/mL Ipilimumab) applied during hospital infusion. After two days from dilution, the protein exhibited aggregation and chemical modifications including oxidation and deamidation. When stability conditions were compromised, the viability of human cell lines treated with the stressed formulation slight decreased suggesting low potential biological toxicity of the modified mAb. As this study has demonstrated the susceptibility of Ipilimumab to light, specific solutions, and excipients as well as the use of safe light in manufacturing, handling, and storage of this drug should be promoted. Moreover, the use of proper primary and secondary packaging should be indicated to avoid the detrimental effect of light on the mAb structure and efficacy. A detailed understanding of Ipilimumab physicochemical properties, integrity, and stability could assure the best storage and manipulation conditions for its safe and successful application in cancer therapy.

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment.

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

Insight into the molecular mechanism underlying the inhibition of α-synuclein aggregation by hydroxytyrosol.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly people. To date, drugs able to reverse the disease are not available; the gold standard is levodopa that only relieves clinical symptoms, yet with severe side effects after prolonged administration. Many efforts are underway to find alternative targets for PD prevention or treatment, the most promising being α-synuclein (Syn). Recently, we reported that oleuropein aglycone (OleA) interferes with amyloid aggregation of Syn both stabilizing its monomeric state and inducing the formation of harmless, off-pathway oligomers. This study is focused at describing the interaction between Syn and hydroxytyrosol (HT), the phenolic moiety and main metabolite of OleA, and the interferences with Syn aggregation by using biophysical and biological techniques. Our results show that HT dose-dependently inhibits Syn aggregation and that covalent and non-covalent binding mediate HT-Syn interaction. HT does not modify the natively unfolded structure of Syn, rather, it stabilizes specific regions of the molecule leading to inhibition of protein fibrillation. Cellular assays showed that HT reduces the toxicity of Syn aggregates. Moreover, Syn aggregates interaction with the cell membrane, an important factor for prion-like properties of Syn on-pathway oligomers, was reduced in cells exposed to Syn aggregates grown in the presence of HT.

Oleuropein aglycone stabilizes the monomeric α-synuclein and favours the growth of non-toxic aggregates.

α-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD); its deposits are found as amyloid fibrils in Lewy bodies and Lewy neurites, the histopathological hallmarks of PD. Amyloid fibrillation is a progressive polymerization path starting from peptide/protein misfolding and proceeding through the transient growth of oligomeric intermediates widely considered as the most toxic species. Consequently, a promising approach of intervention against PD might be preventing α-synuclein build-up, misfolding and aggregation. A possible strategy involves the use of small molecules able to slow down the aggregation process or to alter oligomer conformation favouring the growth of non-pathogenic species. Here, we show that oleuropein aglycone (OleA), the main olive oil polyphenol, exhibits anti-amyloidogenic power in vitro by interacting with, and stabilizing, α-synuclein monomers thus hampering the growth of on-pathway oligomers and favouring the growth of stable and harmless aggregates with no tendency to evolve into other cytotoxic amyloids. We investigated the molecular basis of such interference by both biophysical techniques and limited proteolysis; aggregate morphology was monitored by electron microscopy. We also found that OleA reduces the cytotoxicity of α-synuclein aggregates by hindering their binding to cell membrane components and preventing the resulting oxidative damage to cells.

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids in vitro.

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.

The biological activities of protein/oleic acid complexes reside in the fatty acid.

A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

α-Lactalbumin forms with oleic acid a high molecular weight complex displaying cytotoxic activity.

α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.

The oleic acid complexes of proteolytic fragments of alpha-lactalbumin display apoptotic activity.

The complexes formed by partially folded human and bovine alpha-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine alpha-lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53-103 and the two-chain fragment species 1-40/53-123 and 1-40/104-123, these last being the N-terminal fragment 1-40 covalently linked via disulfide bridges to the C-terminal fragment 53-123 or 104-123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of alpha-helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein-OA complex does not imply specific binding of the protein.

Identification of the core structure of lysozyme amyloid fibrils by proteolysis.

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

A highly amyloidogenic region of hen lysozyme.

Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis.

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.

Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis.

The partly folded states of protein members of the lysozyme (LYS)/alpha-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123- to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far- and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the beta-subdomain (approximately chain region 34-57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured alpha-domain and a largely disrupted beta-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat, trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis.

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.