Phuong Trang Nguyen

Delineating the Role of Helical Intermediates in Natively Unfolded Polypeptide Amyloid Assembly and Cytotoxicity

Amyloid deposition is a hallmark of many diseases, such as the Alzheimers disease. Numerous amyloidogenic proteins, including the islet amyloid polypeptide (IAPP) associated with type II diabetes, are natively unfolded and need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate self-assembly. Recent studies have indicated that the formation of α-helical intermediates accelerates fibrillization, suggesting that these species are on-pathway to amyloid assembly. By identifying an IAPP derivative with a restricted conformational ensemble that co-assembles with IAPP, we observed that helical species were off-pathway in homogenous environment and in presence of lipid bilayers or glycosaminoglycans. Moreover, preventing helical folding potentiated membrane perturbation and IAPP cytotoxicity, indicating that stabilization of helical motif(s) is a promising strategy to prevent cell degeneration associated with amyloidogenesis.

New insights into the roles of sulfated glycosaminoglycans in islet amyloid polypeptide amyloidogenesis and cytotoxicity

Glycosaminoglycans (GAGs) are found in association with virtually all extracellular protein deposits related to amyloid diseases. Particularly, GAGs were shown to enhance fibrillogenesis of the islet amyloid polypeptide (IAPP), a peptide hormone whose aggregation is associated with Type-II diabetes pathogenesis. However, the exact molecular mechanism by which GAGs enhance IAPP amyloidogenesis remains unclear as well as the implications of cell surface GAGs in IAPP-mediated cytotoxicity. The aim of this study was to gain conformational and thermodynamics insights about GAGs-IAPP interactions as a function of IAPP protonation state and buffer ionic strength as well as to explore the roles of cell surface GAGs in IAPP cytotoxicity. Isothermal titration calorimetry revealed that protonation of residue His(18) increases the binding affinity of IAPP towards heparin and, in turn, strongly stimulates fibrillogenesis. Interaction of IAPP with heparin induces a random coil to helix conformational conversion and the helical intermediates could be on-pathway to amyloid fibrils formation. Using rat beta-cells INS-1 that were enzymatically treated with GAG lyases and a CHO cell line that is deficient in the biosynthesis of GAGs, we observed that the lack of GAGs at the plasma membrane does not prevent IAPP-induced toxicity, whereas the presence of soluble heparin in the cell media inhibits IAPP cytotoxicity. Overall, this study reinforces the postulate that sulfated GAGs are actively implicated in IAPP amyloidogenic process in vivo, where they could play a protective role by interacting with cytotoxic species and converting them into less culprit amyloid fibrils.

Secondary conformational conversion is involved in glycosaminoglycans-mediated cellular uptake of the cationic cell-penetrating peptide PACAP

Glycosaminoglycans (GAGs) contribute to the cellular uptake of cationic cell‐penetrating peptides (CPPs). However, molecular details about the contributions of GAGs in CPP internalization remain unclear. In this study, we examined the cellular uptake mechanism of the arginine‐rich CPP pituitary adenylate‐cyclase‐activating polypeptide (PACAP). We observed that the uptake efficacy of PACAP is dependent on the expression of cell surface GAGs. As the binding of PACAP to sulfated GAGs induced a random coil‐to‐α‐helix conformational conversion, we investigated the role of the helical formation in PACAP internalization. Whereas this secondary structure was not crucial for efficient internalization in GAGs‐deficient cells, PACAP α‐helix was essential for GAGs‐dependent uptake.

Mechanistic Contributions of Biological Cofactors in Islet Amyloid Polypeptide Amyloidogenesis

Type II diabetes mellitus is associated with the deposition of fibrillar aggregates in pancreatic islets. The major protein component of islet amyloids is the glucomodulatory hormone islet amyloid polypeptide (IAPP). Islet amyloid fibrils are virtually always associated with several biomolecules, including apolipoprotein E, metals, glycosaminoglycans, and various lipids. IAPP amyloidogenesis has been originally perceived as a self-assembly homogeneous process in which the inherent aggregation propensity of the peptide and its local concentration constitute the major driving forces to fibrillization. However, over the last two decades, numerous studies have shown a prominent role of amyloid cofactors in IAPP fibrillogenesis associated with the etiology of type II diabetes. It is increasingly evident that the biochemical microenvironment in which IAPP amyloid formation occurs and the interactions of the polypeptide with various biomolecules not only modulate the rate and extent of aggregation, but could also remodel the amyloidogenesis process as well as the structure, toxicity, and stability of the resulting fibrils.

Probing the role of λ6 immunoglobulin light chain dimerization in amyloid formation.

Light chain amyloidosis (AL) is a lethal disease associated with the deposition of misfolded immunoglobulin light chains (LC) as amyloid fibrils in the extracellular space of vital organs. The exact mechanisms of LC self-assembly and the molecular basis leading to cellular and organ failure still remain poorly understood. In this study, we investigated the relationship between the quaternary structure, the stability and the amyloidogenecity of LC variable domain (VL) from the λ6 germline. We observed that the amyloidogenic λ6 Wil and its non-amyloidogenic counterpart Jto dimerize in a concentration-dependent manner and that the dimer affinity is considerably decreased in the presence of a high ionic strength. Our results showed that the dimeric state delays the structural conversion associated with amyloid formation and that the monomer is critical to initiate amyloidogenesis. Thermal and chemical unfolding studies revealed that the dimeric state of VL λ6 has an equivalent stability to the monomer. This indicates that the protective effect of dimerization is not related to thermodynamic stability but, most likely, resides in specific structural features. The toxicity of monomeric Jto and Wil as well as fibrillar aggregates was evaluated on cardiomyoblasts and ThT-negative proteospecies reduced cellular viability when employed at high concentration. This study provides novel insights into the complex process of LC amyloidogenesis and suggests that dimer stabilization constitutes a promising strategy to prevent self-assembly and amyloid deposition.

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Role of Site-Specific Asparagine Deamidation in Islet Amyloid Polypeptide Amyloidogenesis: Key Contributions of Residues 14 and 21

Deamidation of an asparagine residue is a spontaneous non-enzymatic post-translational modification that results in the conversion of asparagine into a mixture of aspartic acid and isoaspartic acid. This chemical conversion modulates protein conformation and physicochemical properties, which could lead to protein misfolding and aggregation. In this study, we investigated the effects of site-specific Asn deamidation on the amyloidogenicity of the aggregation-prone peptide islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptidic hormone whose deposition as insoluble amyloid fibrils is closely associated with type 2 diabetes. Asn residues were successively substituted with an Asp or isoAsp, and amyloid formation was evaluated by a thioflavin T fluorescence assay, circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy. Whereas deamidation at position 21 inhibited IAPP conformational conversion and amyloid formation, the N14D mutation accelerated self-assembly and led to the formation of long and thick amyloid fibrils. In contrast, IAPP was somewhat tolerant to the successive deamidation of Asn residues 22, 31, and 35. Interestingly, a small molar ratio of IAPP deamidated at position 14 promoted the formation of nucleating species and the elongation from unmodified IAPP. Besides, using the rat pancreatic β cell line INS-1E, we observed that site-specific deamidation did not significantly alter IAPP-induced toxicity. These data indicate that Asn deamidation can modulate IAPP amyloid formation and fibril morphology and that the site of modification plays a critical role. Above all, this study reinforces the notion that IAPP amyloidogenesis is governed by precise intermolecular interactions involving specific Asn side chains.

Low generation anionic dendrimers modulate islet amyloid polypeptide self-assembly and inhibit pancreatic β-cell toxicity

The deposition of the islet amyloid polypeptide (IAPP) as insoluble amyloid fibrils in the pancreatic islets is associated with type II diabetes. Recent studies have revealed that pre-fibrillar proteospecies and/or the amyloidogenic process mediate β-cell degeneration whereas amyloid fibrils are poorly cytotoxic. Thus, therapeutic strategies that aim at preventing β-cell death associated with amyloid deposition should either sequester prefibrillar species and/or modulate the initial steps of fibrillization. In this view, low generation flexible dendritic scaffolds harboring 4 to 16 hydroxyl, amine, carboxylate or sulfate functional groups were designed and evaluated for their effects on IAPP self-assembly and cytotoxicity. Whereas neutral polyhydroxylated and polycationic dendrimers did not affect the kinetics of amyloid assembly, carboxylated dendrimers accelerated IAPP fibrillization proportionally to surface group density. Interestingly, as revealed by thioflavin T fluorescence, circular dichroism spectroscopy and atomic force microscopy, the G0 sulfated dendrimer inhibited amyloid formation by maintaining the peptide in a random coil conformation. In contrast, G1 sulfated dendrimers potentiated IAPP self-assembly into long amyloid fibrils by a scaffold-based mechanism. Anionic dendrimers attenuated IAPP-induced toxicity on pancreatic β-cells. Our results indicate that sulfated dendrimers can alter the fibrillization pathway of IAPP and inhibit its proteotoxicity, either by accelerating amyloid formation or by trapping the peptide in a non-aggregating and non-toxic state. This study offers novel mechanistic insights for the design of a nanomolecular scaffold to manipulate the self-assembly of natively disordered amyloidogenic peptides.

Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

Over the last two decades, the potential usage of cell-penetrating peptides (CPPs) for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38) has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH) superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs). Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

Current and evolving approaches for improving the oral permeability of BCS Class III or analogous molecules

Abstract The Biopharmaceutics Classification System (BCS) classifies pharmaceutical compounds based on their aqueous solubility and intestinal permeability. The BCS Class III compounds are hydrophilic molecules (high aqueous solubility) with low permeability across the biological membranes. While these compounds are pharmacologically effective, poor absorption due to low permeability becomes the rate-limiting step in achieving adequate bioavailability. Several approaches have been explored and utilized for improving the permeability profiles of these compounds. The approaches include traditional methods such as prodrugs, permeation enhancers, ion-pairing, etc., as well as relatively modern approaches such as nanoencapsulation and nanosizing. The most recent approaches include a combination/hybridization of one or more traditional approaches to improve drug permeability. While some of these approaches have been extremely successful, i.e. drug products utilizing the approach have progressed through the USFDA approval for marketing; others require further investigation to be applicable. This article discusses the commonly studied approaches for improving the permeability of BCS Class III compounds.