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.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

Thiol compounds inhibit the formation of amyloid fibrils by beta 2-microglobulin at neutral pH

Dialysis-related amyloidosis frequently develops in patients undergoing long-term hemodialysis, in which the major component of fibrils is β₂-microglobulin (β2-m). To prevent the disease, it is important to stop the formation of fibrils. β2-m has one disulfide bond, which stabilizes the native structure, and amyloid fibrils. Here, the effects of reductants (i.e., dithiothreitol and cysteine) on the formation of β2-m amyloid fibrils were examined at neutral pH. Fibrils were generated by three methods: seed-dependent, ultrasonication-induced, and salt-and-heat-induced fibrillation. Thioflavin T fluorescence, electron microscopy, and far-UV circular dichroism revealed that the addition of reductants significantly inhibits seed-dependent and ultrasonication-induced fibrillation. For salt-and-heat-induced fibrillation, where the solution of β2-m was strongly agitated, formation of amyloid fibrils was markedly reduced in the presence of reductants, although a small number of fibrils formed even after the reduction of the disulfide bond. The results suggest that reductants such as cysteine and dithiothreitol would be useful for preventing the formation of β2-m amyloid fibrils under physiological conditions.     

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.     

Kinetic refolding barrier of guanidinium chloride denatured goose delta-crystallin leads to regular aggregate formation

Delta-crystallin is the major soluble protein in avian eye lenses with a structural role in light scattering. Dissociation and unfolding of the tetrameric protein in guanidinium chloride (GdmCl) can be sensitively monitored by the intrinsic tryptophan fluorescence. In this study refolding of GdmCl-denatured delta-crystallin was investigated. A marked hysteresis was observed while refolding by dilution of the 5 M GdmCl-denatured delta-crystallin. The secondary structure of the refolded protein was largely restored. However, monitoring intrinsic fluorescence of single tryptophan mutants indicated that the microenvironment of domain 1 (W74) was not restored. The region containing W169, which is close to the dimer interface, remained exposed following refolding. During refolding of the wild-type protein, dimeric, tetrameric, and aggregate forms were identified. The ratio of tetramer to dimer increased with time, as judged by gel-filtration chromatography and nondenaturing gel electrophoresis. However the observed levels of tetramer did not return to the same levels as observed before GdmCl treatment. The proportion of tetramer was significantly decreased in the N-25 deletion mutant and it did not increase with time. These results suggest that there is a kinetic barrier for assembly of dimers into tetramers. The consequence of this is that dimers refold to form aggregates. Aggregation seems to follow a nucleation mechanism with an apparent reaction order of 4.7+/-0.2, suggesting four or five monomers constitute the core structure of nucleus, which propagate to form high molecular weight aggregates. Addition of alpha-crystallin during refolding prevents aggregation. Thioflavin T and Congo red assays indicated a regular structure for the protein aggregates, which appear as hollow tubules packed into helical bundles. Aggregate formation was protein concentration dependent that progressed via two stages with rate constants of 0.0039+/-0.0006 and 0.00043+/-0.00003 s(-1), respectively. We propose that the N-terminal segment of delta-crystallin plays a critical role in proper double dimer assembly and also in the assembly of nucleus to aggregate formation.     

Role of Small Oligomers on the Amyloidogenic Aggregation Free-Energy Landscape

We combine atomic-force-microscopy particle-size-distribution measurements with earlier measurements on 1-anilino-8-naphthalene sulfonate, thioflavin T, and dynamic light scattering to develop a quantitative kinetic model for the aggregation of β-lactoglobulin into amyloid. We directly compare our simulations to the population distributions provided by dynamic light scattering and atomic force microscopy. We combine species in the simulation according to structural type for comparison with fluorescence fingerprint results. The kinetic model of amyloidogenesis leads to an aggregation free-energy landscape. We define the roles of and propose a classification scheme for different oligomeric species based on their location in the aggregation free-energy landscape. We relate the different types of oligomers to the amyloid cascade hypothesis and the toxic oligomer hypothesis for amyloid-related diseases. We discuss existing kinetic mechanisms in terms of the different types of oligomers. We provide a possible resolution to the toxic oligomer-amyloid coincidence.     

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high beta-content, and proteins with significant native alpha-helical structure show increased beta-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.     

Adenylation-dependent conformation and unfolding pathways of the NAD+-dependent DNA ligase from the thermophile Thermus scotoductus

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.     

Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.     

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.     

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF–OA was prepared and its activity and structural changes were compared with α-LA–OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF–OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF–OA was obtained by heat-treatment at pH 8.0 with LD₅₀ of 4.88, 4.95 and 4.62 μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA–OA. Similar to HAMLET, LF–OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF–OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein–OA complexes.     

Conformational properties of the aggregation precursor state of HypF-N

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.