Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1ß in type 2 diabetes.

Interleukin 1ß (IL-1ß) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1ß. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1ß production in vitro. Processing of IL-1ß initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1ß in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion.

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

Energetic basis on interactions between ferredoxin and ferredoxin NADP+ reductase at varying physiological conditions.

In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP+ reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0-200 mM), and temperatures (19-28 °C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases in the NaCl concentration and pH weakened interprotein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependent enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity.

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms.

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12(undiff)). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.

A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein.

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.

Alternatively folded proteins with unexpected beneficial functions.

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.

Refolding of ribonuclease A monitored by real-time photo-CIDNP NMR spectroscopy.

Photo-CIDNP NMR spectroscopy is a powerful method for investigating the solvent accessibility of histidine, tyrosine and tryptophan residues in a protein. When coupled to real-time NMR, this technique allows changes in the environments of these residues to be used as a probe of protein folding. In this paper we describe experiments performed to monitor the refolding of ribonuclease A following dilution from a high concentration of chemical denaturant. These experiments provide a good example of the utility of this technique which provides information that is difficult to obtain by other biophysical methods. Real-time photo-CIDNP measurements yield residue-specific kinetic data pertaining to the folding reaction, interpreted in terms of current knowledge of the folding of bovine pancreatic ribonuclease A.

Photo-CIDNP experiments with an optimized presaturation pulse train, gated continuous illumination, and a background-nulling pulse grid.

Methods to record chemically induced dynamic nuclear polarization (CIDNP) spectra that are virtually free from background magnetization and avoid the sensitivity loss and subtraction artifacts of difference spectroscopy have been developed. Presaturation by a string of composite pi/2 pulses, each followed by a defocussing field gradient, is analyzed, and guidelines for the optimization of pulse phases and gradient strengths are derived. Subsequent gated illumination during a grid of pi pulses with a prescribed timing causes the background magnetization to vanish at those moments of a pulse sequence when CIDNP magnetization is to be sampled or transferred. By shifting the illumination intervals within such a grid, the sign of the polarizations can be inverted without influencing the development of the background magnetization, allowing a further strong suppression of residual background by a phase cycle. Experimental examples for the application of these methods to more complex CIDNP experiments (1D-CIDNP-COSY, 1D-CIDNP-TOCSY, CIDNP-induced heteronuclear Overhauser effects, water suppression in protein CIDNP) are given.

Protein oligomerization induced by oleic acid at the solid-liquid interface--equine lysozyme cytotoxic complexes.

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein alpha-lactalbumin, known as human alpha-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4-30 lysozyme molecules. Each lysozyme molecule is able to bind 11-48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Can misfolded proteins be beneficial? The HAMLET case.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

alpha-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-lactalbumin on the stationary phase in situ. Because native alpha-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.

Conformational analysis in solution of gastrin releasing peptide.

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.

Multiple subsets of side-chain packing in partially folded states of alpha-lactalbumins.

Photochemically induced dynamic nuclear polarization NMR pulse-labeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human alpha-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the alpha-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and non-native environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.

Rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: applications to real-time protein folding.

We describe the development and application of a novel rapid sample-mixing technique for real-time NMR (nuclear magnetic resonance) spectroscopy. The apparatus consists of an insert inside a conventional NMR tube coupled to a rapid injection syringe outside the NMR magnet. Efficient and homogeneous mixing of solutions in the NMR tube is achieved with a dead time of tens of milliseconds, without modification of the NMR probe or additional hardware inside the magnet. Provision is made for the inclusion of an optical fiber to allow in situ laser irradiation of samples, for example to generate photo-CIDNP (chemically induced dynamic nuclear polarization). An NMR water suppression method has been implemented to allow experiments in H(2)O as well as in deuterated solvents. The performance of the device has been tested and optimized by a variety of methods, including sensitive detection of residual pH gradients and the use of NMR imaging to monitor the extent of mixing in real time. The potential utility of this device, in conjunction with the sensitivity and selectivity of photo-CIDNP, is demonstrated by experiments on the protein hen lysozyme. These measurements involve the direct detection of spectra during real-time refolding, and the use of CIDNP pulse labeling to study a partially unfolded state of the protein under equilibrium conditions. Magnetization transfer from this disordered state to the well-characterized native state provides evidence for the remarkable persistence of nativelike elements of structure under conditions in which the protein is partially denatured and aggregation prone.