An increased numbers of ageing diseases associated with metabolism and neurodegenerative processes are involving more individuals in global population. This is unavoidably associated to increased healthcare costs with a significant economic impact in all countries, thus resulting in the requirement of better understanding the molecular mechanisms of ageing. In general, the mechanisms of cellular ageing, often linked to proteins, are nowadays clearer but more information are still requested to open up the prospect of therapeutic intervention for ageing and age related disease.
The number of Alzheimer's disease patients grows rapidly and globally and the need to find effective treatments for the disease becomes more urgent. Diabetes mellitus is today described as a pandemia affecting more than 300 million people worldwide. Generally, in the blood of healthy persons glycated hemoglobin and albumin are considered the main markers for evaluating glycemic excursion in diabets. In healthy conditions, glycated albumin is found in a range between 1% and 10%, in diabetic patients this percentage increases two or three fold. Albumin is a good model protein because is the most abundant protein in plasma and has antioxidant and binding capacities. In fact, oxidative stress and protein modification are two causes of hyperglycemia associated to glucose-protein binding in diabetes disorders. In these last years, glycated albumin has been studied because could be considered a better and short-term glycemic control state in several diabetes-associated pathologies compared to the glycated hemoglobin.
Furthermore, non-enzymatic glycation of proteins is different from glycosylation because only depends on the exposure of free amino-groups in the partially unfolded polypeptide chain, concentration of the sugar and oxidative conditions. It has been reported that non-enzymatic glycation of several proteins stimulates protein aggregation and amyloid deposition. So, the accumulation of proteins as aggregates or as depositions or inclusions in tissues, cause of neurodegenerative diseases, might be favored after glycation, i.e. in diabetic patients. In addition, non-enzymatic glycation appears to play an essential role in the development of diabetic complications: molecular and cellular alterations can become damaging and pathogenic especially in neurodegenerative diseases or in nephropathy or in retinopathy .
The comprehension of the molecular mechanisms linking glycation, aggregation and oxidation of proteins involved in ageing diseases are exciting aspects to explore. Glycation may then be understood as a dynamic contributor to these multifactorial diseases by promoting, accelerating or stabilizing pathological protein aggregation and inducing responses leading to cell dysfunction, damage and death.
With respect to the proteins, amylin and insulin have complementary actions in regulating nutrient levels in the circulation.
Insulin is a well-known protein. Amylin, a 37-residues peptide hormone that is secreted with insulin from the pancreatic ?-cell, is thus deficient in diabetic people. Amylin readily crosses the blood brain barrier and mediates several activities including improving glucose metabolism, relaxing cerebrovascular structure, modulating inflammatory reaction and perhaps enhancing neural regeneration. Human amylin exhibits physicochemical properties predisposing the peptide hormone to aggregate and form amyloid fibrils. Metabolic disorders and aging promote accumulation of amylin amyloid in cerebrovascular system and gray matter altering microvasculature and tissue structure. Deposits of amylin as amyloid are frequently found in the islets of type 2 diabetic individuals and could represent a visible pathological feature that disrupts islet structure and contributes to the islet dysfunction. The peptide circulates in a nonglycosylated (50%) and a glycosylated form, of which the former is the biological active compound. Amylin replacement could possibly improve glycemic control in some people with diabetes. Hyperamylinemia coincides with hyperinsulinemia, is diabetogenic and affects the cardiovascular system. Furthermore, recent study found that treatment with amylin reduces the Alzheimer pathology and improves cognitive impairment in animal models.
We plan to study in parallel the proteins, amylin and insulin. They were chosen because of their relevance in diabetes, their known tendency to aggregate and presence as glycated species and their potential toxicity as such in diabetes related pathologies.
Therefore, the first aim of the study is the comprehension of the interconnected mechanisms in glycation, aggregation and oxidation of proteins and peptides. The second one is how these mechanisms are correlated with ageing diseases and their complications. The final goal could be the development of monoclonal antibodies for glycated aggregates as new biomarkers.
We would use a multidisciplinary approach which exploits structural, biophysical and cellular techniques: producing two specific glycated polypeptides, insulin and amylin, studying their aggregation pathways and producing protein-specific antibodies which could in the future be exploited as selective biomarkers in ageing diseases.
In particular, the project would consist in applying our scientific approach and our expertise to pathological proteins, also by working in synergy with other groups interested. Our techniques are based on biophysical methods in the field of spectroscopy and microscopy with the newest technologies (see the facilities listed below). To follow aggregation processes, our experimental approach mainly consists in kinetic studies of steady-state fluorescence of intrinsic chromophores (tryptophans) and of specific fluorescent dyes, especially Thioflavin T (ThT) for amyloid aggregates. During the kinetics, conformational and structural changes are monitored with FTIR spectroscopy. Size and morphology of the fibrils and oligomers are detected by AFM. Multiple bioimaging technique allowing exploring aggregates and native molecules in live cells and in tissues by means of 3D and 4D measurements. Confocal microscopy, multiphoton microscopy and atomic force microscopy, together with advanced spectroscopy techniques, can be applied to analyse the protein behaviour during and after glycation and/or aggregation and/or oxidation.
Finally, we could also produce monoclonal antibodies for specific glycated products and/or glycated aggregates. Since the glycated adducts have an intrinsically high structural heterogeneity due to the formation of several intermediates in the glycation reaction, commercially available anti-AGEs antibody are unlikely to be effective and specific for the target proteins used in this project. To overcome this issue, the glycated polypeptides at different stages of the glycation and or aggregation processes of amylin will be purified using high-resolution techniques, and supplied to Abcam (Cambridge, UK) for production of monoclonal antibodies. This company is chosen because of their unique RabMAb technology that supplies antibodies, which combine the superior antigen recognition of a rabbit antibody with the specificity and consistency of monoclonal antibodies. This will lead to high affinity and specificity antibodies for each of the isolated glycated polypeptides and aggregates.
Palermo group’s expertise
The Biophysics research group in Palermo (Molecular Biophysics and Soft Matter group http://fisicaechimica.unipa.it/biophysmol/) has already studied glycation of albumin, bovine as a model and human from healthy and diabetic patients [see my papers in CV], using a protocol for non-enzymatic glycation. Glycation, or Millard reaction, is a slow reaction in which the glucose binds free amino groups to form reversibly a Schiff base product. Its rearrangement is known as Amadori product. Further modifications in these early stage glycation products, such as rearrangement, glycoxidation, polymerization give rise the advanced glycation with formation of AGE products strongly oxidants. So, oxidation and glycation are two of the major non-enzymatic mechanism.
In the last years, we have shown that, after almost three weeks and more of protein incubation with glucose at physiological pH, the protein forms amyloid fibrils (pI of glycated albumin decreases) and as a function of carbohydrates used the aggregates change their structure. For example, short-term incubation (7 days) with D-ribose induces albumin to undergo rapid misfolding and to form globular amyloid like aggregates without secondary structure changes [Y. Wei et al., Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells, BMC Cell Biol. 10 (2009)]. Furthermore, the different structural organizations for HSA incubated with glucose for a long time have been reported: large branched chains of globular aggregates with 20-40 nm of average diameters, bundles of unbranched fibrillar aggregates with 140 nm average lengths and fine amorphous aggregates [N. Sattarahmady et al., Detergency effects of nanofibrillar amyloid formation on glycation of human serum albumin, Carbohydr. Res. 343 (2008), B. Bouma et al., Glycation induces formation of amyloid cross-beta structure in albumin, J. Biol. Chem. 278 (2003)]. Amyloid-like aggregates of glycated BSA are able to induce high cytotoxicity that trigger cell death by activation of cellular signaling cascades [Wei Y. et al., Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells. BMC Cell. Biol. (2009)]. Glycation on cells, as for example monocyte cell lines, heated with in vitro glycated albumin reported differential responses of the cells. In particular, HSA induced an inhibition of the proteosomal activities [P. Rondeau et al., Oxidative stresses induced by glycated human or bovine serum albumins on human monocytes, Free Radic. Biol. Med. 45 (2008)].
Another relevant aspect is that albumin can adopt different conformations depending on the ligand bond to the protein and it has binding sites located in three domains. As a consequence of conformational changes, if glycated albumin doesn’t work as scavenger, the oxidative damages and stresses increase, protein aggregation proceeds and the toxicity in cells for the ROS accumulation can occur. These conformational alterations can imply clinical lacks in the drug binding to the albumin especially in diabetes. For evidencing this behavior, we have also studied the effect of a drug as ketoprofen: it acts as a tryptophan quencher via its interaction with HSA purified from plasma of non diabetic and diabetic patients. More the albumin is glycated more the affinity for ketoprofen is diminished for the reduction in the number of binding sites for the drugs. So, alterations in the binding affinity of albumin could have consequences for the pharmacokinetic and pharmacodynamic properties of drugs [J.Baraka-Vidot et al., Glycation alters ligand-binding, enzymatic and pharmacological properties of Human albumin. Biochemistry, 54 (2015), P. Rondeau et al., Thermal aggregation of glycated bovine serum albumin, Biochim. Biophys. Acta 1804 (2010), N. Sattarahmady, Formation of the molten globule-like state during prolonged glycation of human serum albumin, Biochim. Biophys. Acta 1770 (2007), J.Chamani et al., Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates, J. Colloid Interface Sci. 293 (2006), H.Zoellner et al., Fluorometric and mass spectrometric analysis of nonenzymatic glycosylated albumin, Biochem. Biophys. Res. Commun. 284 (2001)].
Furthermore, if Cys34 binds glucose, albumin loses its scavenging property: oxidized albumin through the methionine oxidation (FTIR 900-1200cm-1 without changes in secondary structure), increases its resistance against the amyloid fibrillation. [G.Sancataldo et al. Oxidation enhances human serum albumin thermal stability and changes the routes of amyloid fibril formation. PLoS ONE, 9 (2014)].
Both the information on glycation, oxidation and aggregation could be relevant in designing therapeutic drugs or markers for diagnostic procedures.