Mazin Magzoub

A Peptidomimetic Approach to Targeting Pre-amyloidogenic States in Type II Diabetes

Protein fiber formation is associated with diseases ranging from Alzheimers to type II diabetes. For many systems, including islet amyloid polypeptide (IAPP) from type II diabetes, fibrillogenesis can be catalyzed by lipid bilayers. Paradoxically, amyloid fibers are beta sheet rich while membrane-stabilized states are alpha-helical. Here, a small molecule alpha helix mimetic, IS5, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP-induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative alpha-helical region of IAPP, revealing that alpha-helical states are on pathway to fiber formation. IAPP is not normally amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.

Concentration-dependent transitions govern the subcellular localization of islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β‐cells. In type II diabetes, IAPP aggregates in a process that is associated with β‐cell dysfunction and loss of β‐cell mass. The relationship between IAPPs conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α‐helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPPs capacity to adopt aggregated membrane‐bound α‐helical, and not β‐sheet, states. Our findings suggest that upon α‐helical mediated oligomerization, IAPP acquires cell‐penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.—Magzoub, M. Miranker, A. D. Concentration‐dependent transitions govern the subcellular localization of islet amyloid polypeptide. FASEB J. 26, 1228‐1238 (2012). www.fasebj.org

Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.

Small molecule screening in context: Lipid-catalyzed amyloid formation

Islet Amyloid Polypeptide (IAPP) is a 37‐residue hormone cosecreted with insulin by the β‐cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemers and Parkinsons) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid‐catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.

DNA-assisted oligomerization of pore-forming toxin monomers into precisely-controlled protein channels

Abstract We have developed a novel approach for creating membrane-spanning protein-based pores. The construction principle is based on using well-defined, circular DNA nanostructures to arrange a precise number of pore-forming protein toxin monomers. We can thereby obtain, for the first time, protein pores with specifically set diameters. We demonstrate this principle by constructing artificial alpha-hemolysin (αHL) pores. The DNA/αHL hybrid nanopores composed of twelve, twenty or twenty-six monomers show stable insertions into lipid bilayers during electrical recordings, along with steady, pore size-dependent current levels. Our approach successfully advances the applicability of nanopores, in particular towards label-free studies of single molecules in large nanoscaled biological structures.

Protein aggregation: p53 succumbs to peer pressure

A small subpeptide from the tumor suppressor p53 is shown to mediate aggregation that is similar to that of amyloids. The result is nucleated assembly by mutant p53 that can abrogate function of wild-type p53 and its two functionally important homologs.

Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

Overexpression of mitochondria‐bound hexokinase II (HKII) in cancer cells plays an important role in their metabolic reprogramming and protects them against apoptosis, thereby facilitating their growth and proliferation. Here, we show that covalently coupling a peptide corresponding to the mitochondrial membrane–binding N‐terminal 15 aa of HKII (pHK) to a short, penetration‐accelerating sequence (PAS) enhances the cellular uptake, mitochondrial localization, and cytotoxicity of the peptide in HeLa cells. Further analysis revealed that pHK‐PAS depolarized mitochondrial membrane potential, inhibited mitochondrial respiration and glycolysis, and depleted intracellular ATP levels. The effects of pHK‐PAS were correlated with dissociation of endogenous full‐length HKII from mitochondria and release of cytochrome c. Of significance, pHK‐PAS treatment of noncancerous HEK293 cells resulted in substantially lower cytotoxicity. Thus, pHK‐PAS effectively disrupted the mitochondria‐HKII association in cancer cells, which led to mitochondrial dysfunction and, finally, apoptosis. Our results demonstrate the potential of the pHK‐PAS cell‐penetrating peptide as a novel therapeutic strategy in cancer.—Woldetsadik, A. D., Vogel, M. C., Rabeh, W. M., Magzoub, M. Hexokinase II–derived cell‐penetrating peptide targets mitochondria and triggers apoptosis in cancer cells. FASEB J. 31, 2168–2184 (2017). www.fasebj.org

Cytotoxicity of prion protein-derived cell-penetrating peptides is modulated by pH but independent of amyloid formation

Prion diseases are associated with conversion of cellular prion protein (PrPC) into an abnormally folded and infectious scrapie isoform (PrPSc). We previously showed that peptides derived from the unprocessed N-termini of mouse and bovine prion proteins, mPrP1-28 and bPrP1-30, function as cell-penetrating peptides (CPPs), and destabilize model membrane systems, which could explain the infectivity and toxicity of prion diseases. However, subsequent studies revealed that treatment with mPrP1-28 or bPrP1-30 significantly reduce PrPSc levels in prion-infected cells. To explain these seemingly contradictory results, we correlated the aggregation, membrane perturbation and cytotoxicity of the peptides with their cellular uptake and intracellular localization. Although the peptides have a similar primary sequence, mPrP1-28 is amyloidogenic, whereas bPrP1-30 forms smaller oligomeric or non-fibrillar aggregates. Surprisingly, bPrP1-30 induces much higher cytotoxicity than mPrP1-28, indicating that amyloid formation and toxicity are independent. The toxicity is correlated with prolonged residence at the plasma membrane and membrane perturbation. Both ordered aggregation and toxicity of the peptides are inhibited by low pH. Under non-toxic conditions, the peptides are internalized by lipid-raft dependent macropinocytosis and localize to acidic lysosomal compartments. Our results shed light on the antiprion mechanism of the prion protein-derived CPPs and identify a potential site for PrPSc formation.

Foldamer-Mediated Structural Rearrangement Attenuates Aβ Oligomerization and Cytotoxicity

The conversion of the native random coil amyloid beta (Aβ) into amyloid fibers is thought to be a key event in the progression of Alzheimers disease (AD). A significant body of evidence suggests that the highly dynamic Aβ oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aβ conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aβ oligomers. A library of oligoquinolines was screened to identify antagonists of Aβ oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aβ fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aβ oligomerization and fibrillation. The antagonist activity of 5 toward Aβ aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aβ. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aβ. 5 effectively rescues neuroblastoma cells from Aβ-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aβ and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.

A Hexokinase II Derived-Cell Penetrating Peptide Targets the Mitochondria and Triggers Apoptosis in Cancer Cells

Most cancers are characterized by a high rate of glycolysis and overexpression of mitochondrial-bound isoforms of hexokinase, an enzyme that phosphorylates glucose in an ATP-dependent manner and this commences the first committed step in glucose metabolism. Type II hexokinase (HK II) plays a paramount role in metabolic reprogramming in tumors and its association with the voltage dependent anion channel (VDAC), a major channel for transport of metabolites and ions across the mitochondrial membrane, inhibits apoptosis in cancer cells and is therefore an important therapeutic target. A peptide corresponding to the mitochondrial membrane-binding N-terminal domain of HK II (pHK II) can potentially compete with the endogenous protein for binding to mitochondria and trigger apoptosis. In vitro studies in HeLa cells showed that coupling of pHK II to a short penetration accelerating sequence (Pas: FFLIPKG) enhances the peptides intracellular delivery and cytosolic release, followed by localization to the mitochondria. Cell viability assays revealed that pHK II-pas was considerably more effective in inhibiting cell growth compared to pHK II alone. Moreover, pHK II-pas displayed an enhanced ability to deplete cellular ATP levels and induce apoptosis. Mitochondrial function analysis showed that exposure to pHK II-pas peptide resulted in a significant decrease in glycolytic capacity and glycolytic reserve, as well as basal oxygen consumption rate (OCR), spare respiratory capacity, and ATP turnover. Importantly, these effects were correlated with HK II release from mitochondria. Thus, the mode of action of pHK II-pas involves release of the HK II protein from the mitochondrial membrane resulting in loss of mitochondrial membrane potential, decreased cellular ATP levels and finally apoptosis. Our results underline the potential of the pHK II-pas cell-penetrating peptide (CPP) as an innovative and effective anti-tumor therapeutic strategy.