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Insulin is a key hormone regulating glucose homeostasis, intimately associated with glycemia and is exposed to glycation by glucose, reducing sugars and other highly reactive carbonyls, particularly in diabetes. Glycation of insulin has been reported to differentially affect protein structure, stability and aggregation depending on the glycating agent and experimental conditions. Under reducing conditions glycation produces higher insulin oligomerization thus accelerating amyloid formation whereas, in non-reducing conditions, glycation inhibits amyloid formation. To better detail the effect of glycation on insulin malfunction and toxicity, we investigated the effect of another glycating agent, the D -ribose. Recently, ribosylation has received great interest due to its role in protein glycation and its consequential effects such as protein aggregation, oxidative stress and cell death. Moreover, unusual high concentration of D -ribose has been detected in the urine of type II diabetics. Our results show that, using ribose, as glycating agent, the insulin conformation is preserved and does not evolve in amyloid aggregates because of the block of the α-helix to β-sheet transition, which initiates the aggregation process, maintaining the protein in a soluble state. At the same time, ribose-glycated insulin strongly affects the cell viability, starting a death pathway consisting in the activation of caspases 9 and 3/7, intracellular ROS production and activation of the transcription factor NF-kB.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TAS) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death

"The description of the fibrillogenesis pathway and the identification of “on-pathway” or “off-pathway”. intermediates are key issues in amyloid research as they are concerned with the mechanism for onset of certain. diseases and with therapeutic treatments. Recent results on the fibril formation process revealed an unexpected. complexity both in the number and in the types of species involved, but the early aggregation events are still. largely unknown, mainly because of their experimental inaccessibility. To provide information on the early stage. events of self-assembly of an amyloidogenic protein, during the so-called lag phase, stopped-flow time-resolved. small angle x-ray scattering (SAXS) experiments were performed. Using a global fitting analysis, the structural. and aggregation properties of the apomyoglobin W7FW14F mutant, which is monomeric and partly folded at. acidic pH but forms amyloid fibrils after neutralization, were derived from the first few milliseconds onward.. SAXS data indicated that the first aggregates appear in less than 20 ms after the pH jump to neutrality and further. revealed the simultaneous presence of diverse species. In particular, wormlike unstructured monomers, very. large assemblies, and elongated particles were detected, and their structural features and relative concentrations. were derived as a function of time on the basis of our model. The final results show that, during the lag phase,. early assembling occurs due to the presence of transient monomeric species very prone to association and. through successive competing aggregation and rearrangement processes leading to coexisting on-pathway and. off-pathway transient species."

The conformational dynamics of beta-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of beta-glycosidase similar to those observed in the absence of detergent.

The dynamic properties of the conformational states co-existing during the acid-induced unfolding of tuna apomyoglobin, a single tryptophan-containing protein, have been investigated simultaneously by frequency domain fluorometry. In the transition region, in the absence of salt, the tryptophanyl fluorescence emission arises from a bimodal lifetime distribution. The pH decrease causes a marked broadening of the short-lived distribution component whereas the other component, i.e. the long-lived one, remains unchanged and represented by a very narrow lifetime distribution whose width is similar to that of the native protein. The broadening of the short-lived distribution component observed on lowering the pH indicated that this component arises from fully unfolded molecules. This was further corroborated by acrylamide quenching studies at acidic pH. The collisional quenching rate constant of the short-lived distribution component, i.e. 8.9 x 10(9) M-1s-1, was found to be similar to that observed for a fully exposed residue. The long-lived distribution component was characterized by a lower collisional quenching rate constant, i.e. 2.3 x 10(9) M-1s-1. This value if compared to that determined for the native apoprotein at neutral pH, i.e. 4.0 x 10(8) M-1s-1, indicates that the native-like structure surviving the acid-induced transition possesses a large molecular flexibility.