Michele F. M. Sciacca

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12–18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Calcium-activated membrane interaction of the islet amyloid polypeptide : Implications in the pathogenesis of type II diabetes mellitus

The role played by Ca(2+) ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca(2+) ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca(2+) ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca(2+) ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of beta-sheet rich hIAPP aggregates.

Self-Assembling Pathway of HiApp Fibrils within Lipid Bilayers

The presence of insoluble fibrillar deposits in the pancreas is one of the most common pathological features in patients affected by diabetes mellitus type II. The constituent of these deposits is the hormone known as islet amyloid polypeptide (IAPP) or amylin, a 37-amino-acid polypeptide cosecreted and costored with insulin by Langherans b-cells. This hormone plays a central role in the regulation of glucose’s homeostasis. Human amylin (hIAPP) forms amyloid fibrils in vitro; on the other hand, rat amylin (rIAPP), which differs from the human variant by only six amino acid residues, does not form fibrils and is not toxic. The dysfunction and death of Langherans b-cells has been attributed to the conversion from soluble monomers of hIAPP to insoluble b-sheet fibrils, which lead to extracellular buildup. However, recent studies have refuted this hypothesis, and it is unclear whether Langherans cell death is due to the fibrils or to simple structures. In any case, there is experimental evidence, recently reviewed by Jayasinghe and Langen, of the crucial involvement of lipid membranes in the fibrillogenic process. The fibrillogenesis process of hIAPP follows a nucleation-dependent mechanism that comprise a first phase (lag time), in which monomers form small, soluble, unstructured aggregates, 16] followed by a second phase, in which unstructured aggregates transform into b-sheet-rich structures that form the mature fibrils through a self-assembling process. Moreover, it has been suggested that the main mechanism for hIAPP bcell cytotoxicity can be ascribed to membrane permeabilization, through the formation of membrane channels by soluble hIAPP or by hIAPP-induced bilayer disruption (detergent like). In this work, by using hIAPP in the presence of 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, we provide evidence that the process of membrane destruction is related to the formation of fibrils. POPC lipid membranes were used both as vesicles (LUVs) and supported lipid bilayers (SLBs) in order to simulate the extracellular membrane. Fluorescence spectroscopy, atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) have been employed to characterize the membranes and the interacting hIAPP-membrane systems. In particular, fluorescence versus time measurements have been used to investigate the leakage of a fluorescent probe from LUVs as a test for aspecific membrane pore formations, the thioflavin T (ThT) assay has been used to test the fibril formation, and AFM has been used to obtain morphological information on the studied systems. Finally, the kinetics of hIAPP adsorption onto SLBs have been studied by using QCMD (see the Supporting Information). In Figure 1, the results of fluorescence versus time experiments of hIAPP and 6-carboxyfluorescein-filled LUVs, are shown (red dots). It can be seen that an increase of fluores-

Membrane interactions and conformational preferences of human and avian prion N-terminal tandem repeats: the role of copper(II) ions, pH, and membrane mimicking environments.

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Extracellular truncated tau causes early presynaptic dysfunction associated with Alzheimer’s disease and other tauopathies

The largest part of tau secreted from AD nerve terminals and released in cerebral spinal fluid (CSF) is C-terminally truncated, soluble and unaggregated supporting potential extracellular role(s) of NH2-derived fragments of protein on synaptic dysfunction underlying neurodegenerative tauopathies, including Alzheimers disease (AD). Here we show that sub-toxic doses of extracellular-applied human NH2tau 26-44 (aka NH2htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating in vivo at AD synapses and secreted into parenchyma- acutely provokes presynaptic deficit in K+-evoked glutamate release on hippocampal synaptosomes along with alteration in local Ca2+ dynamics. Neuritic dystrophy, microtubules breakdown, deregulation in presynaptic proteins and loss of mitochondria located at nerve endings are detected in hippocampal cultures only after prolonged exposure to NH2htau. The specificity of these biological effects is supported by the lack of any significant change, either on neuronal activity or on cellular integrity, shown by administration of its reverse sequence counterpart which behaves as an inactive control, likely due to a poor conformational flexibility which makes it unable to dynamically perturb biomembrane-like environments. Our results demonstrate that one of the AD-relevant, soluble and secreted N-terminally truncated tau forms can early contribute to pathology outside of neurons causing alterations in synaptic activity at presynaptic level, independently of overt neurodegeneration.The largest part of tau secreted from AD nerve terminals and released in cerebral spinal fluid (CSF) is C-terminally truncated, soluble and unaggregated supporting potential extracellular role(s) of NH2 -derived fragments of protein on synaptic dysfunction underlying neurodegenerative tauopathies, including Alzheimer’s disease (AD). Here we show that sub-toxic doses of extracellular-applied human NH2 tau 26-44 (aka NH 2 htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating in vivo at AD synapses and secreted into parenchyma- acutely provokes presynaptic deficit in K+ -evoked glutamate release on hippocampal synaptosomes along with alteration in local Ca2+ dynamics. Neuritic dystrophy, microtubules breakdown, deregulation in presynaptic proteins and loss of mitochondria located at nerve endings are detected in hippocampal cultures only after prolonged exposure to NH 2 htau. The specificity of these biological effects is supported by the lack of any significant change, either on neuronal activity or on cellular integrity, shown by administration of its reverse sequence counterpart which behaves as an inactive control, likely due to a poor conformational flexibility which makes it unable to dynamically perturb biomembrane-like environments. Our results demonstrate that one of the AD-relevant, soluble and secreted N-terminally truncated tau forms can early contribute to pathology outside of neurons causing alterations in synaptic activity at presynaptic level, independently of overt neurodegeneration.

Are fibril growth and membrane damage linked processes? An experimental and computational study of IAPP12–18 and IAPP21–27 peptides

Islet amyloid polypeptide (IAPP) is a 37-residue hormone known to deposit as fibrillar aggregates in pancreatic β-cells of patients affected by T2DM. Although it has been proposed that both the fibrillogenic potential and membrane-activity may play a key role in IAPP cytotoxicity, a direct causative relationship between these two properties has not yet been firmly established. More recently, it has been observed that membrane damage may occur independently from fiber formation of IAPP and that these properties may be encoded by different sequences of IAPP. To further check this hypothesis, the membrane-activity and aggregation properties of the two neutral segments LANFLVH (IAPP12–18) and NNFGAIL (IAPP21–27), that recent theoretical studies have reported to possess the highest and the lowest fibrillogenic potential respectively, have been studied by means of a combined experimental and computational approach. The whole of the results demonstrate that if neutral peptides and lipids are employed, the most fibrillogenic peptide has the lowest membrane damaging effect and vice versa. These findings are expected to contribute to our rational understanding of the factors involved in the formation of amyloidosis and in the mechanisms of peptide-induced membrane damage.

The Role of Calcium, Lipid Membranes and Islet Amyloid Polypeptide in the Onset of Type 2 Diabetes: Innocent Bystanders or Partners in a Crime?

Type II diabetes mellitus (T2DM) is one of the most disabling age-related pathologies in developed countries (1). Its incidence is constantly growing: the number of people affected by T2DM worldwide grew from 28 million cases in 1985 to around 168 million in 2001, with an estimate of 350 million cases in 2030 (2). Despite the intense research efforts focusing on T2DM in the last 20 years, the cause of the underlying pathology is still unclear. Islet Amyloid Polypeptide (IAPP) is an amyloidogenic protein, member of the calcitonin family (3), which is known to be involved in the mechanisms lying at the root of T2DM pathogenesis (4, 5). Indeed, although the correlation between islet β-cell death and amyloid fibril formation is still a matter of debate, recent studies, performed on transgenic mice, show that IAPP aggregation can mediate β-cell failure in T2DM (6). Indeed, despite human and rat variants of IAPP differing by only six amino acids, the two variants behave in a completely different way. In particular, rat IAPP (rIAPP) is unable to form amyloid aggregates under normal conditions (7) and rats do not develop T2DM in analogous conditions. Human IAPP (hIAPP) amyloid aggregates are detectable in pancreas’ extracellular space in 90% of patients (4), while only 10% of patients do not have detectable amyloid deposits. We propose that T2DM etiology might be clarified by studying the close relationships between three apparently independent issues: (i) amyloid toxicity; (ii) genetic factors; and (iii) environmental risk factors related to lifestyle and diet.

Copper(II) and zinc(II) dependent effects on Aβ42 aggregation: a CD, Th-T and SFM study

Aβ aggregation is a central event in Alzheimers disease (AD). In vitro evidence indicates that Aβ aggregation and fibrillogenesis are significantly influenced by the employed experimental conditions. Indeed, although it is widely established that metal ions, such as copper and zinc, have significant effects on the Aβ aggregation process, their actual role in Aβ fibrillogenesis is still debated. In this work the effects of a molar excess of zinc(II) and/or copper(II) ions on the Aβ42 aggregation process and the morphology of the resultant aggregates have been compared in samples exhibiting different initial conformations. CD spectroscopy, Th-T-induced fluorescence and Scanning Force Microscopy (SFM) measurements indicated that both metal ions accelerate the aggregation process, yet significantly affect the morphology of aggregates. In particular, copper(II) ions were the most effective in promoting non-fibrillar, amorphous Aβ aggregates. These results further support the hypothesis that an altered distribution of metal ions in neurons might drive alternative Aβ aggregation pathways.

Phospholipids Critical Micellar Concentrations Trigger Different Mechanisms of Intrinsically Disordered Proteins Interaction with Model Membranes

Amyloidogenic proteins are involved in many diseases, including Alzheimers, Parkinsons, and type II diabetes. These proteins are thought to be toxic for cells because of their abnormal interaction with the cell membrane. Simpler model membranes (LUVs) have been used to study the early steps of membrane-protein interactions and their subsequent evolution. Phospholipid LUVs formed in water solution establish a chemical equilibrium between self-assembled LUVs and a small amount of phospholipids in water solution (CMC). Here, using both experimental and molecular dynamics simulations approach we demonstrate that the insertion of IAPP, an amyloidogenic peptide involved in diabetes, in membranes is driven by free lipids in solution in dynamic equilibrium with the self-assembled lipids of the bilayer. It is suggested that this could be a general mechanism lying at the root of membrane insertion processes of self-assembling peptides.

Amyloid growth and membrane damage: Current themes and emerging perspectives from theory and experiments on Aβ and hIAPP

Alzheimers Disease (AD) and Type 2 diabetes mellitus (T2DM) are two incurable diseases both hallmarked by an abnormal deposition of the amyloidogenic peptides Aβ and Islet Amyloid Polypeptide (IAPP) in affected tissues. Epidemiological data demonstrate that patients suffering from diabetes are at high risk of developing AD, thus making the search for factors common to the two pathologies of special interest for the design of new therapies. Accumulating evidence suggests that the toxic properties of both Aβ or IAPP are ascribable to their ability to damage the cell membrane. However, the molecular details describing Aβ or IAPP interaction with membranes are poorly understood. This review focuses on biophysical and in silico studies addressing these topics. Effects of calcium, cholesterol and membrane lipid composition in driving aberrant Aβ or IAPP interaction with the membrane will be specifically considered. The cross correlation of all these factors appears to be a key issue not only to shed light in the countless and often controversial reports relative to this area but also to gain valuable insights into the central events leading to membrane damage caused by amyloidogenic peptides. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.