The structure of tyrosine-10 favors ionic conductance of Alzheimer's disease-associated full-length amyloid-ß channels.

Amyloid ß (Aß) ion channels destabilize cellular ionic homeostasis, which contributes to neurotoxicity in Alzheimer's disease. The relative roles of various Aß isoforms are poorly understood. We use bilayer electrophysiology, AFM imaging, circular dichroism, FTIR and fluorescence spectroscopy to characterize channel activities of four most prevalent Aß peptides, Aß1-42, Aß1-40, and their pyroglutamylated forms (AßpE3-42, AßpE3-40) and correlate them with the peptides' structural features. Solvent-induced fluorescence splitting of tyrosine-10 is discovered and used to assess the sequestration from the solvent and membrane insertion. Aß1-42 effectively embeds in lipid membranes, contains large fraction of ß-sheet in a ß-barrel-like structure, forms multi-subunit pores in membranes, and displays well-defined ion channel features. In contrast, the other peptides are partially solvent-exposed, contain minimal ß-sheet structure, form less-ordered assemblies, and produce irregular ionic currents. These findings illuminate the structural basis of Aß neurotoxicity through membrane permeabilization and may help develop therapies that target Aß-membrane interactions.

Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin.

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER. The free CdtB subunit, which is only active after holotoxin disassembly, then crosses the ER membrane and enters the nucleus where it generates DNA breaks. We hypothesized that the acidified lumen of the endosomes is responsible for separating CdtA from the CdtB/CdtC heterodimer. To test this prediction, possible acid-induced disruptions to the CDT holotoxin were monitored by size exclusion chromatography and surface plasmon resonance. We found that CDT could not efficiently assemble from its individual subunits at the early endosome pH of 6.3. Partial disassembly of the CDT holotoxin also occurred at pH 6.3, with complete separation of CdtA from an intact CdtB/CdtC heterodimer occurring at both pH 6.0 and the late endosome pH of 5.6. Acidification caused the precipitation of CdtA at pH 6.5 and below, but neither CdtB nor CdtC were affected by a pH as low as 5.2. Circular dichroism further showed that the individual CdtB subunit adopts a different secondary structure as compared to its structure in the holotoxin. We conclude the first stage of CDT disassembly occurs in the early endosomes, where an acid-induced alteration to CdtA releases it from the CdtB/CdtC heterodimer.

Challenges and hopes for Alzheimer's disease.

Recent drug development efforts targeting Alzheimer's disease (AD) have failed to produce effective disease-modifying agents for many reasons, including the substantial presymptomatic neuronal damage that is caused by the accumulation of the amyloid ß (Aß) peptide and tau protein abnormalities, deleterious adverse effects of drug candidates, and inadequate design of clinical trials. New molecular targets, biomarkers, and diagnostic techniques, as well as alternative nonpharmacological approaches, are sorely needed to detect and treat early pathological events. This article analyzes the successes and debacles of pharmaceutical endeavors to date, and highlights new technologies that may lead to the more effective diagnosis and treatment of the pathologies that underlie AD. The use of focused ultrasound, deep brain stimulation, stem cell therapy, and gene therapy, in parallel with pharmaceuticals and judicious lifestyle adjustments, holds promise for the deceleration, prevention, or cure of AD and other neurodegenerative disorders.

Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB5-type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT. The difference has been attributed to a more stable domain assembly for CT as compared to LT, but this explanation has not been directly tested and is arguable as toxin disassembly is an indispensable step in the cellular action of these toxins. We show here that PDI disassembles CT more efficiently than LT, which provides a possible explanation for the greater potency of the former toxin. Furthermore, direct examination of CT and LT domain assemblies found no difference in toxin stability. Using novel analytic geometry approaches, we provide a detailed characterization of the positioning of the A subunit with respect to the B pentamer and demonstrate significant differences in the interdomain architecture of CT and LT. Protein docking analysis further suggests that these global structural differences result in distinct modes of PDI-toxin interactions. Our results highlight previously overlooked structural differences between CT and LT that provide a new model for the PDI-assisted disassembly and differential potency of these toxins.

Effects of Aß-derived peptide fragments on fibrillogenesis of Aß.

Amyloid ß (Aß) peptide aggregation plays a central role in Alzheimer's disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aß fibrillogenesis. Although some Aß-derived peptide fragments suppress Aß fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aß sequence has not been reported. The aim of this work is (a) to identify the region(s) of Aß with highest propensities for aggregation and (b) to use those fragments to inhibit Aß fibrillogenesis. Structural and aggregation properties of the parent Aß1-42 peptide and seven overlapping peptide fragments have been studied, i.e. Aß1-10 (P1), Aß6-15 (P2), Aß11-20 (P3), Aß16-25 (P4), Aß21-30 (P5), Aß26-36 (P6), and Aß31-42 (P7). Structural transitions of the peptides in aqueous buffer have been monitored by circular dichroism and Fourier transform infrared spectroscopy. Aggregation and fibrillogenesis were analyzed by light scattering and thioflavin-T fluorescence. The mode of peptide-peptide interactions was characterized by fluorescence resonance energy transfer. Three peptide fragments, P3, P6, and P7, exhibited exceptionally high propensity for ß-sheet formation and aggregation. Remarkably, only P3 and P6 exerted strong inhibitory effect on the aggregation of Aß1-42, whereas P7 and P2 displayed moderate inhibitory potency. It is proposed that P3 and P6 intercalate between Aß1-42 molecules and thereby inhibit Aß1-42 aggregation. These findings may facilitate therapeutic strategies of inhibition of Aß fibrillogenesis by Aß-derived peptides.

Mutual structural effects of unmodified and pyroglutamylated amyloid ß peptides during aggregation.

Amyloid ß (Aß) peptide aggregates are linked to Alzheimer's disease (AD). Posttranslationally pyroglutamylated Aß (pEAß) occurs in AD brains in significant quantities and is hypertoxic, but the underlying structural and aggregation properties remain poorly understood. Here, the structure and aggregation of Aß1-40 and pEAß3-40 are analyzed separately and in equimolar combination. Circular dichroism data show that Aß1-40 , pEAß3-40 , and their combination assume α-helical structure in dry state and transition to unordered structure in aqueous buffer. Aß1-40 and the 1:1 combination gradually acquire ß-sheet structure while pEAß3-40 adopts an α-helix/ß-sheet conformation. Thioflavin-T fluorescence studies suggest that the two peptides mutually inhibit fibrillogenesis. Fourier transform infrared (FTIR) spectroscopy identifies the presence of ß-turn and α-helical structures in addition to ß-sheet structure in peptides in aqueous buffer. The kinetics of transitions from the initial α-helical structure to ß-sheet structure were resolved by slow hydration of dry peptides by D2 O vapor, coupled with isotope-edited FTIR. These data confirmed the mutual suppression of ß-sheet formation by the two peptides. Remarkably, pEAß3-40 maintained a significant fraction of α-helical structure in the combined sample, implying a reduced ß-sheet propensity of pEAß3-40 . Altogether, the data imply that the combination of unmodified and pyroglutamylated Aß peptides resists fibrillogenesis and favors the prefibrillar state, which may underlie hypertoxicity of pEAß.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase.

Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease. The protective role of PDI in Parkinson's Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

Stability and Conformational Resilience of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an abb'xa' structural organization in which the a and a' domains have CGHC active sites, the b and b' domains are involved with substrate binding, and x is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether this unfolding-refolding cycle is a substrate-induced process or an intrinsic physical property of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal denaturation. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54 °C unfolding transition temperature (Tm) and regained 85% of its native structure after nearly complete thermal denaturation. Oxidized PDI had a lower Tm of 48-50 °C and regained 70% of its native conformation after 75% denaturation. Both reduced PDI and oxidized PDI were functional after refolding from these denatured states. Additional studies documented increased stability of a PDI construct lacking the a' domain and decreased thermal stability of a construct lacking the a domain. Furthermore, oxidation of the a domain limited the ability of PDI to refold. The stability and conformational resilience of PDI are thus linked to both redox-dependent and domain-specific effects. These findings document previously unrecognized properties of PDI and provide insight into the physical foundation of its biological function.

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.

FTIR Analysis of Proteins and Protein-Membrane Interactions.

Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. The sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as 13C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, the structural features of membrane pore forming proteins and peptides, and much more. The purpose of this chapter was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, is also presented.

Membrane Pore Formation by Peptides Studied by Fluorescence Techniques.

Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to produce antimicrobial peptides to kill the pathogens by a similar, membrane perforation mechanism. Furthermore, certain endogenous proteins or peptides kill the parent cells through membrane permeabilization. Analysis of the molecular details of membrane pore formation is often conducted using artificial systems, such as bilayer lipid membranes and synthetic peptides. This chapter describes two fluorescence-based methods to study peptide-induced membrane leakage. One method involves preparation of lipid vesicles loaded with a fluorophore (e.g., calcein or carboxyfluorescein) at a self-quenching concentration. If the externally added peptide forms relatively large pores (≥1 nm in diameter), the fluorophore leaks out and undergoes dequenching, resulting in time-dependent increase in fluorescence. The other method is designed to monitor smaller pores (<1 nm in diameter). It involves preparation of vesicles in a Ca2+-less buffer, containing a Ca2+-dependent fluorophore, such as Quin-2. Removal of external Quin-2 by a desalting column and addition of an appropriate concentration of CaCl2 externally sequesters Quin-2 and Ca2+ ions by the vesicle membrane. Addition of the pore-forming peptide to these vesicles results in membrane permeabilization, Ca2+ influx and binding to Quin-2. In both cases, the kinetics of the increase of fluorescence and its equilibrium levels allow quantitative analysis of the pore formation mechanism.

HSC70 and HSP90 chaperones perform complementary roles in translocation of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol.

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol. Heat shock protein 90 (HSP90) binds to the N-terminal region of CTA1 and facilitates its ER-to-cytosol export by refolding the toxin as it emerges at the cytosolic face of the ER membrane. HSP90 also refolds some endogenous cytosolic proteins as part of a foldosome complex containing heat shock cognate 71-kDa protein (HSC70) and the HSC70/HSP90-organizing protein (HOP) linker that anchors HSP90 to HSC70. We accordingly predicted that HSC70 and HOP also function in CTA1 translocation. Inactivation of HSC70 by drug treatment disrupted CTA1 translocation to the cytosol and generated a toxin-resistant phenotype. In contrast, the depletion of HOP did not disrupt CT activity against cultured cells. HSC70 and HSP90 could bind independently to disordered CTA1, even in the absence of HOP. This indicated HSP90 and HSC70 recognize distinct regions of CTA1, which was confirmed by the identification of a YYIYVI-binding motif for HSC70 that spans residues 83-88 of the 192-amino acid CTA1 polypeptide. Refolding of disordered CTA1 occurred in the presence of HSC70 alone, indicating that HSC70 and HSP90 can each independently refold CTA1. Our work suggests a novel translocation mechanism in which sequential interactions with HSP90 and HSC70 drive the N- to C-terminal extraction of CTA1 from the ER.

Structure of amyloid ß25-35 in lipid environment and cholesterol-dependent membrane pore formation.

The amyloid ß (Aß) peptide and its shorter variants, including a highly cytotoxic Aß25-35 peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular membrane permeabilization. The intrinsic polymorphism of Aß has prevented the identification of the molecular basis of Aß pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca2+-transporting Aß25-35 pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aß25-35 has been elucidated. The data suggest that the membrane-embedded peptide forms 6- or 8-stranded ß-barrel like structures. The 8-stranded barrels may conduct Ca2+ ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aß25-35 pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aß membrane pore formation, which should advance both basic and clinical research on Alzheimer's disease and membrane-associated pathologies in general.

Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin.

Cholera toxin (CT) is composed of a disulfide-linked A1/A2 heterodimer and a ring-like, cell-binding B homopentamer. The catalytic A1 subunit must dissociate from CTA2/CTB5 to manifest its cellular activity. Reduction of the A1/A2 disulfide bond is required for holotoxin disassembly, but reduced CTA1 does not spontaneously separate from CTA2/CTB5: protein disulfide isomerase (PDI) is responsible for displacing CTA1 from its non-covalent assembly in the CT holotoxin. Contact with PDI shifts CTA1 from a protease-resistant conformation to a protease-sensitive conformation, which is thought to represent the PDI-mediated unfolding of CTA1. Based solely on this finding, PDI is widely viewed as an 'unfoldase' that triggers toxin disassembly by unfolding the holotoxin-associated A1 subunit. In contrast with this unfoldase model of PDI function, we report the ability of PDI to render CTA1 protease-sensitive is unrelated to its role in toxin disassembly. Multiple conditions that promoted PDI-induced protease sensitivity in CTA1 did not support PDI-mediated disassembly of the CT holotoxin. Moreover, preventing the PDI-induced shift in CTA1 protease sensitivity did not affect PDI-mediated disassembly of the CT holotoxin. Denatured PDI could still convert CTA1 into a protease-sensitive state, and equal or excess molar fractions of PDI were required for both efficient conversion of CTA1 into a protease-sensitive state and efficient disassembly of the CT holotoxin. These observations indicate the 'unfoldase' property of PDI does not play a functional role in CT disassembly and does not represent an enzymatic activity.

From the Wave Equation to Biomolecular Structure and Dynamics.

The multiscale models for complex chemical systems constitute a powerful computational tool to describe biomolecular structure and dynamics, including enzymatic reactions. Here, the development of this method is presented as a miraculous chain of events, involving astoundingly lucky encounters of brilliant minds such as Planck, Schrödinger, Pauling, Karplus, Levitt, and Warshel.

Membrane Binding and Pore Formation by a Cytotoxic Fragment of Amyloid ß Peptide.

Amyloid ß (Aß) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In the brain tissue, Aß occurs in various forms, including an undecapeptide Aß25-35, which exerts a neurotoxic effect through the mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at the biophysical characterization of membrane binding and pore formation by Aß25-35. Interaction of Aß25-35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aß25-35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca2+ influx, and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide-peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aß25-35 forms the most efficient pores in ß-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to eight peptide units, each containing 6-8 monomers.

Unmodified and pyroglutamylated amyloid ß peptides form hypertoxic hetero-oligomers of unique secondary structure.

Amyloid ß (Aß) peptide plays a major role in Alzheimer's disease (AD) and occurs in multiple forms, including pyroglutamylated Aß (AßpE). Identification and characterization of the most cytotoxic Aß species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Aß species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Aß. As a result, the molecular composition and the structural features of "toxic Aß oligomers" have remained unresolved. Here, we have used a novel approach, hydration from gas phase coupled with isotope-edited Fourier transform infrared (FTIR) spectroscopy, to identify the prefibrillar assemblies formed by Aß and AßpE and to resolve the structures of both peptides in combination. The peptides form unusual ß-sheet oligomers stabilized by intramolecular H-bonding as opposed to intermolecular H-bonding in the fibrils. Time-dependent morphological changes in peptide assemblies have been visualized by atomic force microscopy. Aß/AßpE hetero-oligomers exert unsurpassed cytotoxic effect on PC12 cells as compared to oligomers of individual peptides or fibrils. These findings lead to a novel concept that Aß/AßpE hetero-oligomers, not just Aß or AßpE oligomers, constitute the main neurotoxic conformation. The hetero-oligomers thus present a new biomarker that may be targeted for development of more efficient diagnostic and immunotherapeutic strategies to combat AD.

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation.

Pertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established. We addressed these issues with the use of a surface plasmon resonance system to quantify the cytosolic pool of PTS1 from intoxicated cells. Only 3% of surface-associated PTS1 reached the host cytosol after 3 h of toxin exposure. This represented, on average, 38,000 molecules of cytosolic PTS1 per cell. Cells treated with a proteasome inhibitor contained larger quantities of cytosolic PTS1. Stabilization of the dissociated PTS1 subunit with chemical chaperones inhibited toxin export to the cytosol and blocked PT intoxication. ERAD-defective cell lines likewise exhibited reduced quantities of cytosolic PTS1 and PT resistance. These observations identify the unfolding of dissociated PTS1 as a trigger for its ERAD-mediated translocation to the cytosol.

Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

The insulin receptor (IR) is a (αß)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αß heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning ß-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and ß-subunits are linked via a single disulfide bridge, and the (αß)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular ß-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.