February 2024
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17 Reads
Biophysical Chemistry
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February 2024
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17 Reads
Biophysical Chemistry
January 2024
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4 Reads
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1 Citation
Biophysical Chemistry
April 2023
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24 Reads
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2 Citations
Journal of the American Society for Mass Spectrometry
The aggregation of islet amyloid polypeptide (IAPP) is associated with β-cell dysfunction in type 2 diabetes (T2D) in humans. One possible mechanism of toxicity is the interaction of IAPP oligomers with lipid membranes to disrupt the bilayer integrity and/or homeostasis of the cell. Amino acid sequence variations of IAPPs between species can greatly decrease their propensity for aggregation. For example, human IAPP is toxic to β-cells, but rat and pig IAPP are not. However, it is not clear how these differences affect membrane association. Using native mass spectrometry with lipid nanodiscs, we explored the differences in the association of human, rat, and pig IAPP with lipid bilayers. We discovered that human and rat IAPP bound nanodiscs with anionic dipalmitoyl-phosphatidylglycerol (DPPG) lipids, but pig IAPP did not. Furthermore, human and rat IAPP interacted differently with the membrane. Human IAPP show potential tetramer complexes, but rat IAPP associated with the membrane sequentially. Thus, overall IAPP-bilayer interactions are not necessarily related to disease, but small differences in oligomeric behavior at the membrane may instead play a role.
April 2023
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12 Reads
Biochimica et Biophysica Acta (BBA) - Biomembranes
Human islet amyloid polypeptide (hIAPP, also known as amylin) is a 37 amino acid pancreatic polypeptide hormone that plays a role in regulating glucose levels, but forms pancreatic amyloid in type-2 diabetes. The process of amyloid formation by hIAPP contributes to β-cell death in the disease. Multiple mechanisms of hIAPP induced toxicity of β-cells have been proposed including disruption of cellular membranes. However, the nature of hIAPP membrane interactions and the effect of ions and other molecules on hIAPP membrane interactions are not fully understood. Many studies have used model membranes with a high content of anionic lipids, often POPS, however the concentration of anionic lipids in the β-cell plasma membrane is low. Here we study the concentration dependent effect of Ca2+ (0 to 50 mM) on hIAPP membrane interactions using large unilamellar vesicles (LUVs) with anionic lipid content ranging from 0 to 50 mol%. We find that Ca2+ does not effectively inhibit hIAPP amyloid formation and hIAPP induced membrane leakage from binary LUVs with a low percentage of POPS, but has a greater effect on LUVs with a high percentage of POPS. Mg2+ had very similar effects and the effects of Ca2+ and Mg2+ can be largely rationalized by the neutralization of POPS charge. The implications for hIAPP-membrane interactions are discussed.
January 2023
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53 Reads
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1 Citation
Amyloids are partially ordered, proteinaceous, β‐sheet rich deposits that have been implicated in a wide range of diseases. An even larger set of proteins that do not normally form amyloid in vivo can be induced to do so in vitro. A growing number of structures of amyloid fibrils have been reported and a common feature is the presence of a tightly packed core region in which adjacent monomers pack together in extremely tight interfaces, often referred to as steric zippers. A second common feature of many amyloid fibrils is their polymorphous nature. We examine the consequences of disrupting the tight packing in amyloid fibrils on the kinetics of their formation using the 37 residue polypeptide hormone islet amyloid polypeptide (IAPP, amylin) as a model system. IAPP forms islet amyloid in vivo and is aggressively amyloidogenic in vitro. Six Cryo‐EM structures of IAPP amyloid fibrils are available and in all Gly24 is in the core of the structured region and makes tight contacts with other residues. Calculations using the ff14SBonlysc forcefield in Amber20 show that substitutions with larger amino acids significantly disrupt close packing and are predicted to destabilize the various fibril structures. However, Gly to 2‐amino butyric acid (2‐carbon side chain) and Gly to Leu substitutions actually enhance the rate of amyloid formation. A Pro substitution slows, but does not prevent amyloid formation.
January 2023
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43 Reads
The aggregation of islet amyloid polypeptide (IAPP) is associated with beta-cell dysfunction in type 2 diabetes (T2D) in humans. One possible mechanism of toxicity is the interaction of IAPP oligomers with lipid membranes to disrupt bilayer integrity and/or homeostasis of the cell. Amino acid sequence variations of IAPP between species can greatly decrease their propensity for aggregation. For example, human IAPP is toxic to beta-cells, but rat and pig IAPP are not. However, it is not clear how these differences affect membrane association. Using native mass spectrometry with lipid nanodiscs, we explored the differences in the association of human, rat, and pig IAPP with lipid bilayers. We discovered that human and rat IAPP bound nanodiscs with anionic dipalmitoyl-phosphatidylglycerol (DPPG) lipids, but pig IAPP did not. Furthermore, human and rat IAPP interacted differently with the membrane. Human IAPP shows potential tetramer complexes, but rat IAPP associated with the membrane sequentially. Thus, overall IAPP-bilayer interactions are not necessarily related to disease, but differences in oligomeric behavior at the membrane may instead play a role.
November 2022
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38 Reads
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3 Citations
Analytical Chemistry
Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling.
October 2022
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17 Reads
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1 Citation
Biochemistry
Human islet amyloid polypeptide (hIAPP) plays a role in glucose regulation but forms pancreatic amyloid deposits in type 2 diabetes, and that process contributes to β-cell dysfunction. Not all species develop diabetes, and not all secrete an IAPP that is amyloidogenic in vitro under normal conditions, a perfect correlation currently exists between both. Studies of IAPPs from such organisms can provide clues about the high amyloidogenicity of hIAPP and can inform the design of soluble analogues of hIAPP. Sheep and goat IAPP are among the most divergent from hIAPP, with 13 and 11 substitutions, respectively, including an unusual Tyr to His substitution at the C-terminus. The properties of sheep and goat IAPP were examined in solution and in the presence of anionic vesicles, resulting in no observed amyloid formation, even at increased concentrations. Furthermore, both peptides are considerably less toxic to cultured β-cells than hIAPP. The effect of the Y37H replacements was studied in the context of hIAPP, as was a Y37R substitution. Buffer- and salt-dependent effects were observed. There was little impact on the time to form amyloid in phosphate-buffered saline; however, a significant deceleration was observed in Tris buffer, and amyloid formation was slower in the absence of added salt. The Y37H substitution had little impact on toxicity, while the Y37R replacement led to a 30% decrease in toxicity compared with that of hIAPP. The implications for the amyloidogenicity of hIAPP and the design of soluble analogues of the human peptide are discussed.
October 2022
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26 Reads
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4 Citations
Biochemistry
Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone secreted by the pancreatic β-cells. IAPP plays a role in glycemic regulation, but in the pre-type-2 diabetic state, it aggregates to form an islet amyloid. The process of islet amyloid formation contributes to β-cell dysfunction and disease progression. The features of the IAPP sequence that modulate amyloid formation are still not understood. Human IAPP contains three aromatic residues, F15, F23, and Y37. F15 and Y37 are highly conserved, while F23 is more commonly a Leu or Ile in other species. The role of the aromatic residues in modulating the time course of amyloid formation and the cytotoxicity was examined using aromatic to Leu mutations. All three single and double mutants and the triple mutant were studied. F23 plays a dominant role in both amyloid formation and toxicity. An F15L mutant accelerated amyloid formation, a Y37L mutant had little effect, while an F23L replacement slowed amyloid formation by a factor of 2.6. Double mutants, which contained an F23L replacement, had a larger effect than those that did not, and there are non-additive effects between pairs of aromatic residues. F23 also plays a key role in toxicity. Single or multiple mutants that contain the F23L replacement were noticeably less toxic than the wild-type or mutants which did not include the F23L substitution. In contrast, the F15L mutant was more toxic than the wild-type one. The implications for IAPP amyloid formation and for the design of non-aggregating analogues of IAPP are discussed.
July 2022
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47 Reads
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7 Citations
Diabetologia
Aims/hypothesis The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. Methods Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. Results Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001). Conclusions/interpretation Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes. Graphical abstract
... Aromatic stacking interaction is necessary for the binding of hIAPP with amyloid drugs, as it destabilizes the aggregationprone stacking among the aromatic residues of hIAPP itself. 113,114 The aromatic residues Phe15, His18, Phe23, and Tyr37 are involved in π−π stacking interactions with BN nanoparticles ( Figure 5). Phe23 and Tyr37 in the C-terminal display greater stacking probability with BNNS than Phe15 and His18 in the N-terminal, and the reverse is true for the systems containing BNNTs, validating the aforementioned interaction preference of BN nanoparticles ( Figure S7 of the Supporting Information). ...
October 2022
Biochemistry
... In both T1D and T2D structural defects in the islet vasculature have been observed, including thickening and fragmentation of capillaries and an apparent increase in vascular density (58,59). Moreover, in models of T2D, endothelial cells attain an inflamed, pro-adhesive phenotype (49,55,60,61) that renders them incapable of supporting insulin release (55). In CF, changes in islet microvascular density appear to differ profoundly from what is seen in T1D and T2D. ...
July 2022
Diabetologia
... Over the last decade, several modifications of this method have been developed, which differ in, for instance, cyclodextrin species, vesicle size and centrifugation procedures. Besides methyl-β-cyclodextrin (mβCD) [55,[60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77], hydroxypropyl-α-cyclodextrin (HPαCD) [78][79][80][81][82] and methyl-α-cyclodextrin (mαCD) [83,84] was also used. Mainly aLUVs were produced, but some protocols resulted in fabricating aSUVs [61,63,75,76] and aGUVs [55,67,73,75,80]. ...
September 2021
Langmuir
... In many implementations, RBFE alchemical schemes are limited to R-group transformations involving ligand pairs sharing a common scaffold. 8,47 Except for a few commercial products, [48][49][50] scaffold-hopping RBFE calculations involving cyclization, ring expansion, linking, or any other transformation that necessitate the breaking or the formation of chemical bonds, 51 are not generally supported. ...
May 2021
Journal of Chemical Theory and Computation
... The unfolding profile of the bis-Ru adduct Ub-[Ru(cym)][Ru-(cym)(PTA)] (5 + , m/z 1,839) suggests the presence of both the Met1 and His68 adducts in this form with the structural changes induced at similar collision energies as for the mono-adducts, although slightly higher CCS values were observed probably due to the metal complex(es) bound to Ub. Sequential unfolding of this type was previously observed for Cyt c. [30] However, our observations demonstrate that the identity of the adduct(s) affects the protein ion conformation at low energy, as well as the ion stability and unfolding pathway. A similar trend was observed in recent CIU investigations of partially and hetero-metalated Cd-, Zn-, Cu-and Ag-metallothionein (MT) adducts, [31] where different metals or levels of metalation, e. g., comparing Cd 1 -, Cd 2 -, Zn 1 -, or Cu 3 Cd 1 -MT, resulted in differences in protein compactness, stability, and unfolding pathways. ...
May 2021
Journal of the American Society for Mass Spectrometry
... A more recent paper on a mutant of the L9 protein (CTL9), which undergoes cold denaturation at temperatures above freezing, stated that "the structural properties of pressureunfolded states of proteins do not differ substantially from those of unfolded states at atmospheric pressure and are, if anything, more, rather than less, expanded than their atmospheric state counterparts". 21 This conclusion was based on the comparison between X-ray scattering studies and high-pressure diffusion ordered NMR experiments. ...
May 2021
Biophysical Journal
... In addition to an exploration of the effect of point mutations on already foldedexperimental and predicted-3D structures, MD simulations are used to address the folding process, in spite of Levinthal's paradox [114,[120][121][122]; unfolding [96,97,123]; and dynamics of intrinsically disordered proteins (IDPs) [82,124]. Such tasks particularly highlight the problem of computational cost and conformational sampling in MD simulations [125]. ...
March 2021
The Journal of Physical Chemistry B
... For example, longrange contacts were found to contribute to the intramolecular heterogeneity of the IDP prion monomer, which adopts collapsed and extended regions (28). Recently, it has been shown that, longrange contacts in the cold denatured state made the ensemble contract, deviating from the homopolymer model (29). Similarly, long-range stickers of aromatic residues tend to contract IDPs (30). ...
July 2020
Biochemistry
... Pancreases were extracted and fixed in 10% neutral buffered formalin (SF100-4, ThermoFisher Scientific). Fixed pancreas samples were embedded in paraffin, processed, 4 mm sections were cut, then sections were stained as previously described [19]. Briefly, b cells were stained using mouse monoclonal anti-insulin antibody (I2018; MilliporeSigma) followed by goat anti-mouse Cy3 (115-165-146; Jackson ImmunoResearch, West Grove, PA). ...
November 2020
Diabetologia
... It is one of the most amyloidogenic natural sequences known and forms amyloid even more rapidly than the Aβ peptide of Alzheimer's disease. The sequence of IAPP is highly conserved, however not all species develop type-2 diabetes and those which do not produce and secrete a form of IAPP that is non amyloidogenic in vivo (Noh et al., 2020;Westermark et al., 1990). In contrast, species that develop type-2 diabetes also develop islet amyloid and produce an amyloidogenic version of IAPP. ...
May 2020
ACS Chemical Biology