Nataliya Popovych

Role of zinc in human islet amyloid polypeptide aggregation

Human Islet Amyloid Polypeptide (hIAPP) is a highly amyloidogenic protein found in islet cells of patients with type II diabetes. Because hIAPP is highly toxic to beta-cells under certain conditions, it has been proposed that hIAPP is linked to the loss of beta-cells and insulin secretion in type II diabetics. One of the interesting questions surrounding this peptide is how the toxic and aggregation prone hIAPP peptide can be maintained in a safe state at the high concentrations that are found in the secretory granule where it is stored. We show here zinc, which is found at millimolar concentrations in the secretory granule, significantly inhibits hIAPP amyloid fibrillogenesis at concentrations similar to those found in the extracellular environment. Zinc has a dual effect on hIAPP fibrillogenesis: it increases the lag-time for fiber formation and decreases the rate of addition of hIAPP to existing fibers at lower concentrations, while having the opposite effect at higher concentrations. Experiments at an acidic pH which partially neutralizes the change in charge upon zinc binding show inhibition is largely due to an electrostatic effect at His18. High-resolution structures of hIAPP determined from NMR experiments confirm zinc binding to His18 and indicate zinc induces localized disruption of the secondary structure of IAPP in the vicinity of His18 of a putative helical intermediate of IAPP. The inhibition of the formation of aggregated and toxic forms of hIAPP by zinc provides a possible mechanism between the recent discovery of linkage between deleterious mutations in the SLC30A8 zinc transporter, which transports zinc into the secretory granule, and type II diabetes.

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Background: cytb5 modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb5 was solved by NMR, and the cytP450-binding site on cytb5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb5-cytP450 complex is presented. Addition of a substrate strengthens the cytb5-cytP450 interaction. Significance: The cytb5-cytP450 complex structure will help unravel the mechanism by which cytb5 regulates catalysis by cytP450. Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

Site specific interaction of the polyphenol EGCG with the SEVI amyloid precursor peptide PAP(248-286).

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

NMR Structure in a Membrane Environment Reveals Putative Amyloidogenic Regions of the SEVI Precursor Peptide PAP248−286

Semen is the main vector for HIV transmission worldwide. Recently, a peptide fragment (PAP(248-286)) has been isolated from seminal fluid that dramatically enhances HIV infectivity by up to 4-5 orders of magnitude. PAP(248-286) appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. We have solved the atomic-level resolution structure of the SEVI precursor PAP(248-286) using NMR spectroscopy in SDS micelles, which serve as a model membrane system. PAP(248-286), which does not disrupt membranes like most amyloid proteins, binds superficially to the surface of the micelle, in contrast to other membrane-disruptive amyloid peptides that generally penetrate into the core of the membrane. The structure of PAP(248-286) is unlike most amyloid peptides in that PAP(248-286) is mostly disordered when bound to the surface of the micelle, as opposed to the alpha-helical structures typically found of most amyloid proteins. The highly disordered nature of the SEVI peptide may explain the unique ability of SEVI amyloid fibers to enhance HIV infection as partially disordered amyloid fibers will have a greater capture radius for the virus than compact amyloid fibers. Two regions of nascent structure (an alpha-helix from V262-H270 and a dynamic alpha/3(10) helix from S279-L283) match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. The structure presented here can be used for the rational design of mutagenesis studies on SEVI amyloid formation and viral infection enhancement.

Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1−40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed.

The amyloidogenic SEVI precursor, PAP248-286, is highly unfolded in solution despite an underlying helical tendency

Amyloid fibers in human semen known as SEVI (semen-derived enhancer of viral infection) dramatically increase the infectivity of HIV and other enveloped viruses, which appears to be linked to the promotion of bridging interactions and the neutralization of electrostatic repulsion between the host and the viral cell membranes. The SEVI precursor PAP(248-286) is mostly disordered when bound to detergent micelles, in contrast to the highly α-helical structures found for most amyloid proteins. To determine the origin of this difference, the structures of PAP(248-286) were solved in aqueous solution and with 30% and 50% trifluoroethanol. In solution, pulsed field gradient (PFG)-NMR and (1)H-(1)H NOESY experiments indicate that PAP(248-286) is unfolded to an unusual degree for an amyloidogenic peptide but adopts significantly helical structures in TFE solutions. The clear differences between the structures of PAP(248-286) in TFE and SDS indicate electrostatic interactions play a large role in the folding of the peptide, consistent with the slight degree of penetration of PAP(248-286) into the hydrophobic core of the micelle. This is another noticeable difference between PAP(248-286) and other amyloid peptides, which generally show penetration into at least the headgroup region of the bilayer, and may explain some of the unusual properties of SEVI.

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ~75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ~13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.

General In-Vitro Catalysis of Amyloid Formation by the Bacterial Curli Protein

Proteins misfolded into insoluble, fibrillar aggregates known as amyloid are a pathological feature of many common and devastating diseases. Amyloid formation is typically a slow process that can be strongly affected by extrinsic factors, among the most critical being the presence of a small amount of preformed seeds that serves to nucleate aggregation. Amyloid nucleation is often considered a highly specific process dependent on a high degree of similarity in both peptide sequence and fiber morphology. However, we show here that amyloid fibers known as curli that are produced in E. coli and related bacteria catalyze amyloid formation of a variety of dissimilar amyloidogenic peptides and proteins, including PAP248-286 (SEVI), insulin, and calcitonin. The preformed curli fibers appear to act as a nucleation site for amyloidogenic proteins and as such, can decrease the induction time, sometimes drastically, and induce the formation of fibers. In particular, cross-seeding of SEVI amyloid formation by curli was more effective than seeding the reaction with SEVI amyloid fibers obtained under a different reaction condition. The elongation rate of fiber formation is also increased for some (but not all) of the proteins tested, indicating curli can also increase in some circumstances the rate of addition of proteins to the ends of amyloid fibers. Curli and curli-like amyloid fibers are ubiquitous in mammalian hosts, in fact, the innate immune response common to almost all amyloids has been proposed to have evolved as a response to curli amyloid formation by E. Coli. Given that certain bacteria that express a curli-like protein colocalize with amyloid deposits in Alzheimer?s patients, the induction of amyloid formation by curli may be a factor of high clinical importance.

Clues for the Mechanism of SEVI HIV Enhancement from Structural Studies of the SEVI Precursor Peptide PAP248-286 in a Membrane Environment by NMR

Despite the rapid progress of the AIDS pandemic, the HIV virus is a surprisingly weak pathogen in vitro. The large difference between in vitro and in vivo infection rates suggests that cofactors absent in vitro but essential for the natural transmission of the virus may be responsible for this discrepancy. A recently identified peptide in human semen, PAP248-286, has emerged as a clear candidate for the missing cofactor as it dramatically enhances the infectivity of HIV by up to five orders of magnitude. PAP248-286 appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. To understand the unique ability of SEVI to enhance HIV infection, we have solved the atomic-level resolution structure of the SEVI precursor PAP248-286 using NMR spectroscopy in SDS micelles. In contrast to other toxic amyloid peptides that generally penetrate into the core of the membrane, non-toxic PAP248-286 binds superficially to the surface of the micelle. Unlike most amyloid peptides that bind to the membrane in an α-helical state, PAP248-286 is mostly disordered when bound to the surface of the micelle. The highly disordered nature of the SEVI peptide may explain the high ability of SEVI to enhance HIV infection, as partially disordered amyloid fibers will have a greater capture radius for the virus than more compact amyloid fibers. Two regions of nascent structure match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. NMR studies of the binding of PAP248-286 to the anti-amyloid agent ECGC will also be presented.