Christoph Meier

Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

Naturally Occurring Fragments from Two Distinct Regions of the Prostatic Acid Phosphatase Form Amyloidogenic Enhancers of HIV Infection

ABSTRACT Semen is the major vector for HIV-1 transmission. We previously isolated C-proximal fragments of the prostatic acid phosphatase (PAP) from semen which formed amyloid fibrils that potently enhanced HIV infection. Here, we used the same methodology and identified another amyloidogenic peptide. Surprisingly, this peptide is derived from an N-proximal fragment of PAP (PAP85-120) and forms, similar to the C-proximal fragments, positively charged fibrillar structures that increase virion attachment to cells. Our results provide a first example for amyloid formation by fragments of distinct regions of the same precursor and further emphasize the possible importance of amyloidogenic peptides in HIV transmission.

Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen

Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presence of amyloid fibrils in semen remained to be demonstrated. Here, we use state of the art confocal and electron microscopy techniques for direct imaging of amyloid fibrils in human ejaculates. We detect amyloid aggregates in all semen samples and find that they partially consist of PAP fragments, interact with HIV particles and increase viral infectivity. Our results establish semen as a body fluid that naturally contains amyloid fibrils that are exploited by HIV to promote its sexual transmission.

Surface attachment of protein fibrils via covalent modification strategies.

Chemical control of surface functionality and topography is an essential requirement for many technological purposes. In particular, the covalent attachment of monomeric proteins to surfaces has been the object of intense studies in recent years, for applications as varied as electrochemistry, immuno-sensing, and the production of biocompatible coatings. Little is known, however, about the characteristics and requirements underlying surface attachment of supramolecular protein nanostructures. Amyloid fibrils formed by the self-assembly of peptide and protein molecules represent one important class of such structures. These highly organized beta-sheet-rich assemblies are a hallmark of a range of neurodegenerative disorders, including Alzheimers disease and type II diabetes, but recent findings suggest that they have much broader significance, potentially representing the global free energy minima of the energy landscapes of proteins and having potential applications in material science. In this paper, we describe strategies for attaching amyloid fibrils formed from different proteins to gold surfaces under different solution conditions. Our methods involve the reaction of sulfur containing small molecules (cystamine and 2-iminothiolane) with the amyloid fibrils, enabling their covalent linkage to gold surfaces. We demonstrate that irreversible attachment using these approaches makes possible quantitative analysis of experiments using biosensor techniques, such as quartz crystal microbalance (QCM) assays that are revolutionizing our understanding of the mechanisms of amyloid growth and the factors that determine its kinetic behavior. Moreover, our results shed light on the nature and relative importance of covalent versus noncovalent forces acting on protein superstructures at metal surfaces.

A molecular tweezer antagonizes seminal amyloids and HIV infection

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a ‘molecular tweezer’ specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion–amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses. DOI: http://dx.doi.org/10.7554/eLife.05397.001

Hierarchically self-assembled host-guest network at the solid-liquid interface for single-molecule manipulation

The manufacture of functional molecular devices is one of the key research topics in nanotechnology. For applications in molecular storage and quantum computing, molecules must be arranged in a repetitive structure and also be addressable and manipulable in a controlled fashion. The self-assembly of molecular building blocks with hydrogen-bonding capabilities is a suitable method for generating highly ordered and porous two-dimensional (2D) hydrogen-bonded networks (HBNs). These porous 2D HBNs can be used to immobilize organic and inorganic guest molecules in a spatially wellordered arrangement, predetermined by the host network structure. The controlled manipulation of guest molecules was demonstrated for various functional guest molecules by means of scanning tunneling microscopy (STM) experiments but has been limited so far to the controlled desorption or the lateral manipulation of single molecules. In contrast to ultrahigh-vacuum (UHV) conditions where the reservoir of manipulable molecules is restricted to the number of adsorbed species, the supernatant liquid phase at the solid–liquid interface in principle offers an almost unlimited depot of molecules (“ink”) and is therefore the perfect experimental environment for tip-controlled adsorption of guest molecules into the HBN. The “ink” attribute of a supernatant solution is used in scanning-probe-based lithographic techniques such as replacement lithography and dip-pen lithography to tailor the chemical composition and structure of a surface on the 100 nm scale. So far, these lithographic techniques are limited to a resolution of about 15 nm. For the spatially controlled adsorption of guest molecules in an HBN, the host–guest system must fulfill the following requirements: 1) the host network must be inert towards the manipulation process; 2) the dynamics of the manipulated components must be slow enough in order to follow the result of the manipulation with STM; and 3) the occupation of the cavities with guest molecules should be low such that unoccupied host cavities are available. All of these requirements need well-balanced adsorbate–adsorbate and substrate–adsorbate interactions. Here we present a host–guest network that meets the demands for a spatially tip-controlled single-molecule manipulation. After describing the outstanding properties of our host–guest system, we demonstrate the spatially tip-controlled desorption of guest molecules from the cavities of the host network. Moreover, we show for the first time the tipcontrolled adsorption of single solvated guest molecules at the solid–liquid interface. The C2v-symmetric HBN building block 3,3’-BTP forms a polymorphic supramolecular HBN on highly ordered pyrolytic graphite (HOPG). The porous 2D network was used to generate a hierarchically self-assembled host–guest architecture with copper(II) phthalocyanine (CuPc) as guest molecule. The occupation of individual HBN cavities with CuPc can be altered with a voltage pulse applied to the tip (“erasing” and “writing”). As we recently

pH Responsive Janus-like Supramolecular Fusion Proteins for Functional Protein Delivery

A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins β-galactosidase and the enzymatic subunit of Clostridium botulinum C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques.

Conducting core-shell nanowires by amyloid nanofiber templated polymerization.

The preparation of conducting polymer nanowires in aqueous solutions is a challenging goal, especially for applications in nanobioelectronics. Here, we show that amyloid nanofibers template the formation of conducting polyaniline nanowires with a core-shell architecture. The nanofibers exhibit hydrophobic pockets that presumably preassemble the aniline monomers. The template directs polymer morphology as it favors the formation of linear polymer chains, suppresses defects in the polymer chain which are detrimental to charge transport and induces chiral helicity into the polymer. This strategy has the potential of being applied to other polymers than polyaniline and might open up new possibilities to synthesize biocompatible and conducting polymer nanowires with prospects for applications in, for example, sensing, neuronal tissue engineering, and electrostimulated stem cell differentiation.

Peptide nanofibrils as enhancers of retroviral gene transfer

Amyloid fibrils are polypeptide-based polymers that are typically associated with neurodegenerative disorders such as Alzheimers disease. More recently, it has become clear that amyloid fibrils also fulfill functional roles in hormone storage and biosynthesis. Furthermore, it has been demonstrated that semen contains abundant levels of polycationic amyloid fibrils. The natural role of these seminal amyloids remains elusive. Strikingly, however, they drastically enhance HIV-1 infection and may be exploited by the virus to increase its sexual transmission rate. Their strong activity in enhancing HIV-1 infection suggests that seminal amyloid might also promote transduction by retroviral vectors. Indeed, SEVI (semen-derived enhancer of virus infection), the best characterized seminal amyloid, boosts retroviral gene transfer more efficiently than conventional additives. However, the use of SEVI as laboratory tool for efficient retroviral gene transfer is limited because the polypeptide monomers are relatively expensive to produce. Furthermore, standardized production of SEVI fibrils with similar high activities is difficult to achieve because of the stochastic nature of the amyloid assembly process. These obstacles can be overcome by recently identified smaller peptides that spontaneously self-assemble into nanofibrils. These nanofibrils increase retroviral gene transfer even more efficiently than SEVI, are easy to produce and to handle, and seem to be safe as assessed in an ex vivo gene transfer study. Furthermore, peptide-based nanofibrils allow to concentrate viral particles by low-speed centrifugation. Specific adaption and customization of self-assembling peptides might lead to novel nanofibrils with versatile biological functions, e.g., targeted retroviral gene transfer or drug delivery.

Self-Assembly of High Molecular Weight Polypeptide Copolymers Studied via Diffusion Limited Aggregation

The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells.