Dieter Willbold

The N-Terminus of Nef from HIV-1/SIV Associates with a Protein Complex Containing Lck and a Serine Kinase

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.

Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy

Dual-focus fluorescence correlation spectroscopy is a method for precisely measuring the diffusion coefficient of fluorescing molecules close to the infinite dilution limit in a reference-free and absolute manner. We apply the method to determine the diffusion coefficients of three fluorescent dyes across the visible spectrum. These values can be used as absolute reference standards for fluorescence correlation spectroscopy. In particular, it is found that the diffusion coefficient of the widely used reference dye Rhodamine 6G is by 37% larger than the value used in most publications on fluorescence correlation spectroscopy over the last three decades.

Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy.

Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The γ-aminobutyric acid type A (GABAA) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pulldown analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

Pyroglutamate Formation Influences Solubility and Amyloidogenicity of Amyloid Peptides

N-Terminally truncated and pyroglutamate (pGlu) modified amyloid beta (Abeta) peptides are major constituents of amyloid deposits in sporadic and inherited Alzheimers disease (AD). Formation of pGlu at the N-terminus confers resistance against cleavage by most aminopeptidases, increases toxicity of the peptides, and may seed Abeta aggregate formation. Similarly, the deposited amyloid peptides ABri and ADan, which cause a very similar histopathology in familial British dementia (FBD) and familial Danish dementia (FDD), are N-terminally blocked by pGlu. Triggered by the coincidence of pGlu-modified amyloid peptides and similar pathology in AD, FBD, and FDD, we investigated the impact of N-terminal pGlu on biochemical and biophysical properties of Abeta, ABri, and ADan. N-Terminal pGlu increases the hydrophobicity and changes the pH-dependent solubility profile, rendering the pGlu-modified peptides less soluble in the basic pH range. The pGlu residue increases the aggregation propensity of all amyloid peptides as evidenced by ThT fluorescence assays and dynamic light scattering. The far-UV CD spectroscopic analysis points toward an enhanced beta-sheet structure of the pGlu-Abeta. Importantly, changes in fibril morphology are clearly caused by the N-terminal pGlu, resulting in the formation of short fibers, which are frequently arranged in bundles. The effect of pGlu on the morphology is virtually indistinguishable between ABri, ADan, and Abeta. The data provide evidence for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD. The main driving force for the aggregation is apparently an increase in the hydrophobicity and thus an accelerated seed formation.

Selection of D-Amino-Acid peptides that bind to Alzheimer's disease amyloid peptide A beta(1-42) by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimers disease amyloid peptide Aβ(1–42). A randomized 12‐mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Aβ(1–42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D‐pep) was shown to bind Aβ(1–42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimers disease, amyloid plaques and leptomeningeal vessels containing Aβ amyloid were stained specifically with a fluorescence‐labeled derivative of D‐pep. Fibrillar deposits derived from other amyloidosis were not labeled by D‐pep. Possible applications of this novel and highly specific Aβ ligand in diagnosis and therapy of Alzheimers disease are discussed.

Integral Membrane Proteins in Nanodiscs Can Be Studied by Solution NMR Spectroscopy

We present a two-dimensional solution NMR spectrum of an integral membrane protein (IMP) in a nanodisc. Solution NMR relies on rapid isotropic tumbling of the analyte with correlation times in the nanosecond range. IMPs in a cellular membrane do not satisfy this condition. Previous liquid-state NMR studies on IMPs were conducted in organic solvent or artificial membrane mimicking particles like detergent micelles. Nanodiscs are relatively small (150 kDa), detergent-free model membranes that are suitable for functional reconstitution of IMPs. Nanodiscs allow solubilization of integral membrane proteins in a nearly native lipid bilayer environment. The 70 residue polypeptide CD4mut was incorporated into nanodiscs. CD4mut features one transmembrane helix. The aliphatic (1)H-(13)C HSQC spectrum of nanodiscs with inserted, ((13)C, (15)N)-labeled CD4mut exhibits reasonably dispersed protein and lipid NMR signals. Our results demonstrate that IMPs in nanodiscs are amenable to liquid-state NMR methodology.

Fibril structure of amyloid-beta (1-42) by cryo-electron microscopy.

Elucidating pathological fibril structure Amyloid-β (Aβ) is a key pathological contributor to Alzheimers disease. Gremer et al. used cryoelectron microscopy data to build a high-quality de novo atomic model of Aβ fibrils (see the Perspective by Pospich and Raunser). The complete structure reveals all 42 amino acids (including the entire N terminus) and provides a structural basis for understanding the effect of several disease-causing and disease-preventing mutations. The fibril consists of two intertwined protofilaments with an unexpected dimer interface that is different from those proposed previously. The structure has implications for the mechanism of fibril growth and will be an important stepping stone to rational drug design. Science, this issue p. 116; see also p. 45 Cryo–electron microscopy structure of an amyloid-β(1–42) fibril reveals a protofilament interface and the entire N-terminal region. Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer’s disease patients. We present the structure of an Aβ(1–42) fibril composed of two intertwined protofilaments determined by cryo–electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-β structure resulting in an overall “LS”-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed “groove” and “ridge,” leading to different binding pathways on both fibril ends, which has implications for fibril growth.

HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins

The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential resonance assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.

Reduction of Alzheimer's disease amyloid plaque load in transgenic mice by D3, A D-enantiomeric peptide identified by mirror image phage display.

Alzheimer’s disease (AD) is a multifactorial disorder, which is characterized by progressive memory deficits, cognitive impairments and personality changes. More than 20 million people are affected worldwide. The histopathological hallmarks of AD are aggregated protein deposits (i.e. , senile plaques and neurofibrillary tangles) in the brain. Senile plaques consist mainly of extracellular amyloid-b peptide (Ab) deposits. While there is still debated over whether Ab is the causative agent in AD, the inhibition of Ab production and aggregation is often targeted for therapy development. Recently, we used mirror image phage display to identify a novel d-amino acid peptide binding specifically to Ab ACHTUNGTRENNUNG(1–42) with a binding affinity in the submicromolar dissociation constant range and called it “d-pep” or “D1”. 3] d-peptides are known to be extremely protease resistant and less immunogenic than their respective l-enantiomers, thus being more suitable for in vivo use. d-Peptides have previously been used as inhibitors of amyloid formation to prevent the associated Ab cytotoxicity. Recently, another strategy to obtain d-peptides specifically interacting with amyloid stretches, inhibiting amyloid formation and cell toxicity, was presented. In the present work, we identified a novel d-enantiomeric amino acid peptide “D3” which might provide a novel basis for therapeutic and preventive approaches to AD. D3 might also be useful as a tool to study the role of Ab plaques in AD progression. We performed phage display selections of a peptide library encoding more than 1 10 randomly different 12amino acid sequences with d-enantiomeric Ab ACHTUNGTRENNUNG(1–42) (d-Ab) as the target. d-Ab was dissolved to obtain a low final concentration of 2 nm. Under those conditions, we expected monomeric Ab or small Ab oligomers to be the dominant target species during the phage display screening. After 6 rounds of biopanning, we determined the peptide sequences of the enriched phage displayed peptides by DNA sequence analysis of the respective genome region. The dominant peptide sequence obtained from the selection was RPRTRLHTHRNR, referred to as D3. This sequence was found in 9 out of 23 randomly chosen phage clones. Additionally, 9 of the selected peptides were related to the dominating peptide by at least 9 amino acids (table S1, Supporting Information). We investigated the influence of D3 on Ab aggregation. The content of amyloid fibrils was determined by Thioflavin T (ThT) fluorescence upon incubation of various Ab/D3 mixtures. ThT is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils, and is commonly used to detect amyloid fibrils. ThT fluorescence of Ab mixtures with D3 was significantly lower than those without D3 (Figure 1 a). These results suggest that D3 significantly decreased the formation of ThT-positive Ab aggregates. These results were confirmed using a fluorescence correlation spectroscopy (FCS) based assay. FCS allows the detection of Ab aggregates in highly dilute samples, with concentrations in the nanomolar range. Fluorescence fluctuations generated by single molecules passing the confocal volume of the focused laser beam are measured, and can be evaluated by autocorrelation to obtain the diffusion time of the studied molecules. When Ab aggregates, containing at least one molecule of Oregon Green (OG) labeled Ab, are present within a given solution, they can be directly detected as spikes in the fluorescence fluctuation recordings. Due to their size, they have an increased duration of stay in the confocal volume and increased fluorescence intensities because most or all of them contain more than one fluorescence label. FCS measurements were carried out with 5 nm OG-labeled Ab in the absence or presence of D3 in various concentrations. Figure 1 b shows how the number of peaks decreases with increasing amounts of added D3. A 50 % inhibition of aggregate formation by D3 is obtained at ~1 mm. This result suggests that D3 prevents aggregation of Ab in the nanomolar concentration range. To assay the ability of D3 to dissolve pre-existing ThT positive Ab aggregates, Ab was preincubated without D3 for seven days to allow aggregation. Then, D3 at various concentrations was added and ThT fluorescence was followed (Figure 1 c). The results clearly show a dose-dependent aggregate disassembly activity of D3 for preformed ThT positive Ab aggregates without stirring, ultrasonic treatment, or any other mechanical support. We studied the effect of D3 on Ab-induced cytotoxicity in rat pheochromocytoma (PC12) cells. Ab (10 mm) was incubated without or with varying concentrations of D3 for 6 days at 37 8C. Cells were then treated with various Ab/D3 mixtures and their viability was measured by MTT reduction (Figure 1 d). In the presence of 2 mm of Ab cell viability dropped to 40 %, this effect was reversed in a dose-dependant manner by the addition of D3, and cell viability could be completely rescued in [a] Dr. K. Wiesehan, Dr. S. A. Funke, Prof. Dr. D. Willbold INB-2/Molekulare Biophysik II Forschungszentrum J lich, 52425 J lich (Germany) Fax: (+ 49) 2461612233 E-mail : d.willbold@fz-juelich.de [b] Dr. L. Nagel-Steger, Prof. Dr. D. Willbold Institut f r Physikalische Biologie, BMFZ, Heinrich-Heine-Universit t 40225 D sseldorf (Germany) [c] Dr. T. van Groen, Dr. I. Kadish Dept. Cell Biology, University of Alabama at Birmingham, AL 35294 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.200800273.