Protein biochemistry

• Amyloid


  Protein aggregation and deposition associated with several diseases
• Natively unfolded proteins


  Proteins that need to undergo conformational rearrangements to form amyloids
• Intrinsically disordered polypeptides (IDPs)


  Proteins like amyloid-β and islet amyloid polypeptide (IAPP) that need to form locally ordered structures to self-assemble
• Intermediate species of the amyloidogenic cascade are more deleterious than fibrils
• IAPP


  The main component of amyloid deposits in pancreatic islets of type II diabetes patients
• Two models for IAPP conformational rearrangements that initiate oligomerization and amyloid formation


  • Formation of β-strand rich dimers and assembly from ordered β-hairpins
  • Association thermodynamically linked to helix formation within the 5-22 segment

  
• Helical intermediates


  Proposed to be important for the aggregation and toxicity of amyloidogenic IDPs
• Detecting transient intermediate species of the amyloidogenic cascade is challenging, particularly in the context of interactions with biological factors
• [f15, l16]hIAPP (D-hIAPP)


  IAPP derivative with a restricted conformational ensemble that co-assembles with IAPP
• Incorporation of two D-residues within the putative helical region of IAPP


  Prevents TFE-induced helical folding
• 1. hIAPP and hIAPP have equivalent kinetics of self-assembly into amyloid fibrils
• Fibrils formed by D-hIAPP and hIAPP are different at the macroscopic level but have comparable secondary structure

  
• Equimolar mixture of hIAPP and D-hIAPP has equivalent kinetics of amyloid formation as homogenous solutions
• Incorporation of D-residues in non-amyloidogenic rodent IAPP decreases its helical folding but does not affect its amyloidogenicity
• Binding of IAPP to anionic membranes or glycosaminoglycans


  Facilitates IAPP adopting a helical conformation before converting into β-sheets
• Hindering the helical folding of IAPP


  Dramatically hastens its self-assembly into amyloids in the presence of membranes or glycosaminoglycans
• Preventing helical folding potentiates membrane perturbation and IAPP cytotoxicity
• q220/q208 ratio


  Correlated closely to the kinetics of amyloid formation measured by ThT-fluorescence

  
• Preventing helical folding potentiated the amyloidogenic effect of heparin, employed as a model of the sulfated domains of heparan sulfate
• Random coil-to-a-helix conformational conversion


  Occurs upon binding of IAPP to anionic biosurfaces
• Inhibition of helical formation dramatically hastens self-assembly of IAPP
• Helical intermediates are off-pathway to amyloid formation
• Protein deposition in pancreatic islets


  • Correlates with b-cell degeneration
  • Several inter-connected mechanisms have been proposed to explain IAPP toxicity
• Plasma membrane disruption by pre-fibrillar species


  Can trigger various pathways of cell death
• Loss of membrane integrity results from pore formation and helical intermediates could be the active membrane species
• Several helical mimetics were recently developed to target the membrane-bound a-helix and to prevent their oligomerization
• These helical mimetics were shown to reduce IAPP amyloid formation in presence of membranes and to decrease toxicity
• Whether or not these helical species are toxic themselves or are off- or on-pathway to cytotoxicity remains unknown
• The helically-frustrated D-hIAPP analog appears as a unique tool to address this question
• Peptides hIAPP and D-hIAPP induced a similar concentration-dependant vesicle leakage


  Non-amyloidogenic rIAPP and D-rIAPP also induced membrane leakage, although their effects were less pronounced
• Time course of vesicle disruption by hIAPP


  1. Plateau after 60 min followed by a second phase leading to maximum leakage

  2. Second phase correlated closely with formation of ThT-positive species and the kinetics of a-helix-to-b-sheet structural conversion
• For the non-amyloidogenic rodent IAPPs, which do not form ThT-positive species in presence of LUVs, a single phase was detected in the time course of vesicle disruption

  
• The first phase of membrane disruption is related to the binding of non-fibrillar species to lipid vesicles and the second phase is mediated by fibril growth
• The multistep process was not discernable for D-hIAPP, because fibrillization occurs so fast that it cannot be easily discernible from prefibrillar species binding
• Vesicle leakage experiments indicated that IAPP helical folding postpones membrane disruption associated with fibril growth
• Incorporation of a destabilizing motif within hIAPP putative a-helix

  Increased its cytotoxicity
• 1. hIAPP was not only more cytotoxic


  Its deleterious effects on viability occurred more rapidly
• After 12 h, hIAPP (50 μM) reduced cellular viability to 63.8 ± 9.0% while a viability of 12.4 ± 2.1% was observed for its d,d-counterpart
• A significant increase of apoptosis signal was detected after only 4 h treatment with D-hIAPP whereas a treatment time of 12 h was needed to activate caspases with hIAPP
• Similar effect was observed for cytosolic calcium level, a signal of cellular dysfunction associated with several death mechanisms