HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Nanomechanical properties of SSTSAA microcrystals are dominated by the inter-sheet packing.

Amyloid fibrils have been associated with human disease for many decades, but it has also become apparent that they play a functional, non-disease-related role in e.g. bacteria and mammals. Moreover, they have been shown to possess interesting mechanical properties that can be harnessed for future man-made applications. Here, the mechanical behaviour of SSTSAA microcrystals has been investigated. The SSTSAA peptide organization in these microcrystals has been related to that in the corresponding amyloid fibrils. Using high-pressure X-ray diffraction experiments, the bulk modulus K, which is the reciprocal of the compressibility ß, has been calculated to be 2.48 GPa. This indicates that the fibrils are tightly packed, although the packing of most native globular proteins is even better. It is shown that the value of the bulk modulus is mainly determined by the compression along the c-axis, that relates to the inter-sheet distance in the fibrils. These findings corroborate earlier data obtained by AFM and molecular dynamics simulations that showed that mechanical resistance varies according to the direction of the applied strain, which can be related to packing and hydrogen bond contributions. Pressure experiments provide complementary information to these techniques and help to acquire a full mechanical characterization of biomolecular assemblies. This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 2)'.

Insects as an Alternative Source for the Production of Fats for Cosmetics.

Insects may provide an environmentally friendly way of producing high-quality bio-based materials that can be implemented for cosmetic applications. Insects can be bred on organic waste, in high numbers, and on small surfaces, therefore, making large scale industrial breeding possible. Fats from three insect species: the black soldier fly (BSF) (Hermetia illucens), the locust (Locusta migratoria), and the house cricket (Acheta domesticus) were evaluated for potential use in skin care. Insects were dried and fats were extracted using petroleum ether. The fats were further refined, and the fatty acid composition and the acid value were determined. The fats were used in a hand cream formulation and compared with the currently used mink-and plant-derived oils. Fatty acid analysis indicates that BSF contains > 60% of lauric acid, which makes it less suitable for application in a skin-care product, whereas locust and cricket fats are rich in C16 and C18 fatty acids which makes them more suitable. Phospholipids and free fatty acid levels in the three insect species are relatively high compared with commercial, refined oils, and need to be removed by appropriate refining protocols. Odor and color also need to be removed by physical refinement to improve the applicability.

In-situ spectroscopic investigation of ultrasonic assisted unfolding and aggregation of insulin.

It is well-known that fibrillogenesis of proteins can be influenced by diverse external parameters, such as temperature, pressure, agitation or chemical agents. The present preliminary study suggests that ultrasonic excitation at moderate intensities has a significant influence on the unfolding and aggregation behaviour of insulin. Irradiation with an average sound intensity of even as low as 70mW/cm(2) leads to a lowering of the unfolding and aggregation temperature up to 7K. The effect could be explained by an increase of the aggregation kinetics due to ultrasonically induced acoustic micro-streaming in the insulin solution that most probably enhances the aggregation rate. The clear and remarkable effect at relatively low sound intensities offers interesting options for further applications of ultrasound in biophysics and biochemistry. On the other hand, a process that causes a change of kinetics equivalent to 7K also gives a warning signal concerning the safety of those medical ultrasonic devices that work in this intensity range.

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin.

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV-vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60°C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80°C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal ß-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.

Catalytic and chaperone-like functions in an intrinsically disordered protein associated with desiccation tolerance.

Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure.

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the ß-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter- and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.

FTIR 2D correlation spectroscopy of α1 and α2 fractions of an alkali-pretreated gelatin.

An alkali-pretreated gelatin (pI~4.9) was fractionated by means of alcohol coacervation and semi-preparative gel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatin were investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600-1700 cm⁻¹). The gelation temperature was the same for all examined samples (24.5°C) while the melting temperature of the α2 fraction was lower (30°C) than that of the other samples (32.5°C). The 2D COS plots indicate that on cooling (gelation) the core sequence of conformational changes is the same for all samples. On heating, however, the α2 fraction deviates from the α1-containing samples and shows an earlier disappearance of the triple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of the α2 fraction thus results from a different conformational cascade of the α2 chains upon melting. In all samples the initial conformational changes take place in the ß-turns, providing further evidence for the models proposed previously.

Structural and mechanical properties of TTR105-115 amyloid fibrils from compression experiments.

Amyloid fibrils, originally associated with neurodegenerative diseases, are now recognized to have interesting mechanical properties. By using synchrotron x-ray diffraction at high pressure in a diamond anvil cell we determined the bulk modulus of TTR105-115 amyloid fibrils in water and in silicone oil to be 2.6 and 8.1 GPa, respectively. The compression characteristics of the fibrils are quite different in the two media, revealing the presence of cavities along the axis of the fibrils, but not between the ß-sheets, which are separated by a dry interface as in a steric zipper motif. Our results emphasize the importance of peptide packing in determining the structural and mechanical properties of amyloid fibrils.

Nanomechanical and structural properties of native cellulose under compressive stress.

Cellulose is an important biopolymer with applications ranging from its use as an additive in pharmaceutical products to the development of novel smart materials. This wide applicability arises in part from its interesting mechanical properties. Here we report on the use of high pressure X-ray diffraction and Raman spectroscopy in a diamond anvil cell to determine the bulk and local elastic moduli of native cellulose. The modulus values obtained are 20 GPa for the bulk modulus and 200-355 and 15 GPa for the crystalline parts and the overall elastic (Young's) modulus, respectively. These values are consistent with those calculated from tensile measurements. Above 8 GPa, the packing of the cellulose chains within the fibers undergoes significant structural distortion, whereas the chains themselves remain largely unaffected by compression.

An X-ray and neutron scattering study of the equilibrium between trimethylamine N-oxide and urea in aqueous solution.

The interaction of the osmolytes trimethylamine N-oxide (TMAO) and urea in aqueous solutions at 40 °C was investigated by isotopic substitution neutron scattering at a TMAO mole fraction of 0.05 and TMAO/urea concentration ratios of 1 : 2 and 1 : 4. The partial pair distribution functions obtained by the empirical potential structure refinement method are consistent with those obtained previously for similar pure TMAO and 1 : 1 TMAO-urea solutions and indicate that urea progressively replaces the water molecules in the first coordination shell of the TMAO oxygen atom. The apparent association constant for the TMAO : urea complex (K(1)) was calculated to be 0.14 M(-1), which is of the same order as the experimental urea-protein binding constants per site reported in the literature. This confirms that the two osmolytes act independently at least in the physiological range.

The Right-Handed Parallel ß-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure.

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-ß pectinolytic enzyme with a right-handed parallel ß-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol-1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner.

Consistent picture of the reversible thermal unfolding of hen egg-white lysozyme from experiment and molecular dynamics.

Synchrotron radiation circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopies, and small-angle x-ray scattering were used to monitor the reversible thermal unfolding of hen egg white lysozyme. The results were compared with crystal structures and high- and low-temperature structures derived from molecular-dynamics calculations. The results of both experimental and computational methods indicate that the unfolding process starts with the loss of ß-structures followed by the reversible loss of helix content from ∼40% at 20°C to 27% at 70°C and ∼20% at 77°C, beyond which unfolding becomes irreversible. Concomitantly there is a reversible increase in the radius of gyration of the protein from 15 Å to 18 Å. The reversible decrease in forward x-ray scattering demonstrates a lack of aggregation upon unfolding, suggesting the change is due to a larger dilation of hydration water than of bulk water. Molecular-dynamics simulations suggest a similar sequence of events and are in good agreement with the (1)H(N) chemical shift differences in nuclear magnetic resonance. This study demonstrates the power of complementary methods for elucidating unfolding/refolding processes and the nature of both the unfolded structure, for which there is no crystallographic data, and the partially unfolded forms of the protein that can lead to fibril formation and disease.

Effect of enzymatic deimination on the conformation of recombinant prion protein.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

Counteraction of urea by trimethylamine N-oxide is due to direct interaction.

Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins, induces folding, and counteracts the denaturing effects of urea, pressure, and ice. To establish the mechanism behind these effects, isotopic substitution neutron-scattering measurements were performed on aqueous solutions of TMAO and 1:1 TMAO-urea at a solute mole fraction of 0.05. The partial pair distribution functions were extracted using the empirical potential structure refinement method. The results were compared with previous results obtained with isosteric tert-butanol, as well as the available data from spectroscopy and molecular-dynamics simulations. In solution, the oxygen atom of TMAO is strongly hydrogen-bonded to, on average, between two and three water molecules, and the hydrogen-bond network is tighter in water than in pure water. In TMAO-urea solutions, the oxygen atom in TMAO preferentially forms hydrogen bonds with urea. This explains why the counteraction is completed at a 2:1 urea/TMAO concentration ratio, independently of urea concentration. These results strongly support models for the effect of TMAO on the stability of proteins based on a modification of the simultaneous equilibria that control hydrogen bonding between the peptide backbone and water or intramolecular sites, without any need for direct interaction between TMAO and the protein.

A generic mechanism of emergence of amyloid protofilaments from disordered oligomeric aggregates.

The presence of oligomeric aggregates, which is often observed during the process of amyloid formation, has recently attracted much attention because it has been associated with a range of neurodegenerative conditions including Alzheimer's and Parkinson's diseases. We provide a description of a sequence-indepedent mechanism by which polypeptide chains aggregate by forming metastable oligomeric intermediate states prior to converting into fibrillar structures. Our results illustrate that the formation of ordered arrays of hydrogen bonds drives the formation of beta-sheets within the disordered oligomeric aggregates that form early under the effect of hydrophobic forces. Individual beta-sheets initially form with random orientations and subsequently tend to align into protofilaments as their lengths increase. Our results suggest that amyloid aggregation represents an example of the Ostwald step rule of first-order phase transitions by showing that ordered cross-beta structures emerge preferentially from disordered compact dynamical intermediate assemblies.

In Vivo Water Dynamics in Shewanella oneidensis Bacteria at High Pressure.

Following observations of survival of microbes and other life forms in deep subsurface environments it is necessary to understand their biological functioning under high pressure conditions. Key aspects of biochemical reactions and transport processes within cells are determined by the intracellular water dynamics. We studied water diffusion and rotational relaxation in live Shewanella oneidensis bacteria at pressures up to 500 MPa using quasi-elastic neutron scattering (QENS). The intracellular diffusion exhibits a significantly greater slowdown (by -10-30%) and an increase in rotational relaxation times (+10-40%) compared with water dynamics in the aqueous solutions used to resuspend the bacterial samples. Those results indicate both a pressure-induced viscosity increase and slowdown in ionic/macromolecular transport properties within the cells affecting the rates of metabolic and other biological processes. Our new data support emerging models for intracellular organisation with nanoscale water channels threading between macromolecular regions within a dynamically organized structure rather than a homogenous gel-like cytoplasm.

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

Thermal stability of homologous functional units of Helix pomatia hemocyanin does not correlate with carbohydrate content.

The thermal stability of the eight functional units of beta-hemocyanin of the gastropodan mollusc Helix pomatia was investigated by FTIR spectroscopy. Molluscan hemocyanin functional units have a molecular mass of approximately 50 kDa and generally contain three disulfide bridges: two in the mainly alpha-helical N-terminal domain and one in the C-terminal beta-sheet domain. They show more than 50% sequence homology and it is assumed that they adopt a similar conformation. However, the functional units of H. pomatiabeta-hemocyanin, designated HpH-a to HpH-h, differ considerably in their carbohydrate content (0-18 wt%). Most functional units are exceptionally stable with a melting temperature in the range 77-83 degrees C. Two functional units, HpH-b and HpH-c, however, have a reduced stability with melting temperature values of 73 degrees C and 64 degrees C, respectively. Although the most glycosylated functional unit (HpH-g) has the highest temperature stability, there is no linear correlation between the degree of glycosylation of the functional units and the unfolding temperature. This is ascribed to variations in secondary structure as well as in glycan attachment sites. Moreover, the disulfide bonds might play an important role in the conformational stability of the functional units. Sequence comparison of molluscan hemocyanins suggests that the less stable functional units, HpH-b and HpH-c, similar to most of their paralogous counterparts, lack the disulfide bond in the C-terminal domain.

Phase behavior of N-(Isopropyl)propionamide in aqueous solution and changes in hydration observed by FTIR spectroscopy.

The phase behavior of N-(isopropyl)propionamide (NiPPA), which is the repeat unit of poly(N-isopropyl-acrylamide) (PNiPA), in deuterated aqueous solution was investigated. Temperature induces a phase separation of NiPPA in aqueous solution above the lower critical solution temperature (LCST), as shown by optical microscopy. The phase behavior of NiPPA resembles that of PNiPA, but the demixing domain is much narrower. Monitoring the liquid-liquid phase separation by Fourier transform infrared (FTIR) spectroscopy reveals that a fraction of the NiPPA molecules becomes dehydrated above the LCST. Our findings therefore shed new light on the results of a recent dielectric relaxation experiment in which the behavior of NiPPA was found to be "completely contrary" to that of PNiPA. It is argued that the differences in the spectroscopic results of polymer and repeat unit solutions can be easily understood from the phase behavior of NiPPA and PNiPA.