Jeannine Mohrlüder

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.

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4‐Aminobutyrate type A (GABAA) receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in the intracellular trafficking of GABAA receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra‐Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca2+‐dependent chaperone and a Ca2+‐buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra‐endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull‐down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant Kd = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.

Structure and potential function of γ‐aminobutyrate type A receptor‐associated protein

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier, and is implicated in a variety of membrane trafficking and fusion events that are crucial to synaptic plasticity, autophagy and apoptosis. However, important aspects of GABARAP function and regulation remain poorly understood. We review the current state of knowledge about GABARAP, highlighting newly‐identified GABARAP ligands, and discuss the possible physiological relevance of each ligand interaction.

Structural framework of the GABARAP–calreticulin interface – implications for substrate binding to endoplasmic reticulum chaperones

The 4‐aminobutyrate type A receptor‐associated protein (GABARAP) is a versatile adaptor protein that plays an important role in intracellular vesicle trafficking, particularly in neuronal cells. We have investigated the structural determinants underlying the interaction of GABARAP with calreticulin using spectroscopic and crystallographic techniques. Specifically, we present the crystal structure of GABARAP in complex with its major binding epitope on the chaperone. Molecular modeling of a complex containing full‐length calreticulin suggests a novel mode of substrate interaction, which may have functional implications for the calreticulin/calnexin family in general.

Ligand binding mode of GABAA receptor-associated protein.

The gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA(A) receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 A resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA(A) receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.

An indole binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP

The role of tryptophan as a key residue for ligand binding to the ubiquitin‐like modifier GABAA receptor associated protein (GABARAP) was investigated. Two tryptophan‐binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified. The two tryptophan binding sites can be clearly distinguished by mapping the NMR spectroscopy‐derived residue‐specific apparent dissociation constant, Kd, onto the three‐dimensional structure of GABARAP. The biological relevance of tryptophan‐binding pockets of GABARAP was supported by a highly conserved tryptophan residue in the GABARAP binding region of calreticulin, clathrin heavy chain, and the gamma2 subunit of the GABAA receptor. Replacement of tryptophan by alanine abolished ligand binding to GABARAP.

Interaction of Bcl-2 with the Autophagy-related GABAA Receptor-associated Protein (GABARAP) BIOPHYSICAL CHARACTERIZATION AND FUNCTIONAL IMPLICATIONS

Background: Apoptosis and autophagy are coordinately regulated, but the underlying mechanisms are incompletely understood. Results: Bcl-2 specifically interacts with GABARAP via a conserved EWD motif, resulting in impaired GABARAP lipidation. Conclusion: Sequestration of GABARAP is likely to contribute to the down-regulation of autophagy by Bcl-2. Significance: Interfering with pro-survival functions of Bcl-2 (including its impact on autophagy) represents a promising strategy for cancer therapy. Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.

Comparative modeling of human NSF reveals a possible binding mode of GABARAP and GATE‐16

Vesicular trafficking is an important homeostatic process in eukaryotic cells which critically relies on membrane fusion. One of the essential components of the universal membrane fusion machinery is NSF (N‐ethylmaleimide‐sensitive factor), a large hexameric ATPase involved in disassembly of SNARE (soluble NSF attachment protein receptor) complexes. To improve our understanding of this sophisticated molecular machine, we have modeled the structure of the NSF hexamer in two alternative assemblies. Our data suggest a mechanistic concept of the operating mode of NSF which helps to explain the functional impact of post‐translational modifications and mutations reported previously. Furthermore, we propose a binding site for the ubiquitin‐like proteins GABARAP and GATE‐16, which is supported by experimental evidence, yielding a complex with favorable surface complementarity. Proteins 2009.

Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8(G116) shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

Preparation of a functional GABARAP-lipid conjugate in nanodiscs and its investigation by solution NMR spectroscopy.

Membrane proteins are essential for many physiological processes and are of particular importance as targets in modern drug discovery. Knowledge about their 3D fold is crucial for an improved understanding of associated biological events on a molecular level. An important challenge for functional and structural studies of peripheral and integral membrane proteins is the development of a suitable membrane-mimicking environment that supports the structural and functional integrity of the protein of interest. For liquid-state NMR investigations of membrane proteins, detergent micelles or detergent/ lipid bicelles are the most commonly used membrane mimetics. Unfortunately, they often display significant surface curvature of the water/lipid interface, which can affect or even destabilize the structure of the protein. Unfavorable dynamics of membrane proteins in micelles or bicelles furthermore impair the quality of the resulting NMR spectra. A promising model membrane system is provided by nanodisc technology, in which a discoidal lipid bilayer (~160 lipid molecules) is encircled by two copies of an engineered derivative of apolipoprotein A-I, the membrane scaffold protein (MSP). The nanodisc diameter of ~10 nm is defined by the length of MSP and is tunable by design. The particle thickness of ~5 nm and the number of incorporated lipids are lipid dependent. Numerous membrane proteins that exhibit different topologies have been successfully introduced into nanodiscs in their native states. Besides a variety of integral membrane proteins, a small number of peripheral and membrane-tethered proteins were also investigated. While structural aspects of lipidated proteins have been previously investigated in vesicles, no studies using nanodisc-inserted proteolipids have yet been performed. In the current report, we focus on the preparation as well as the structural and functional investigation of a special subgroup of membrane proteins, namely the C-terminally lipidated proteins of the GABARAP-like family. The human GABAA receptor-associated protein (GABARAP) is one of the mammalian homologues of yeast Atg8, which is required for autophagosome formation. Autophagy recruits autophagosomes to degrade bulky proteins and organelles in order to maintain cellular homeostasis. The lipidation and delipidation of Atg8 mediate the tethering, hemifusion, and expansion of autophagosomal membranes in yeast, while its homologues undergo similar processes in mammalian cells. GABARAP is known to be a versatile adaptor protein implicated in vesicular transport events and autophagy. Like other proteins of the GABARAPlike family, the C-terminal residue of fully translated GABARAP is proteolytically cleaved off to yield GABARAP-I, which has then a glycine at its C-terminal end. This glycine (G116) is subsequently enzymatically coupled to the phospholipid phosphatidylethanolamine (PE) or phosphatidylserine (PS) to generate GABARAP-II. 14] GABARAP-II delipidation is also enzyme mediated and recycles GABARAP-I. The modification state of GABARAP is proposed to be crucial for its intracellular distribution, which is relevant for its function during autophagy. GABARAP consists of 117 amino acids and has a molecular weight of about 14 kDa. While the structure of nonlipidated GABARAP has already been solved, that of lipidated GABARAP has yet to be elucidated. In order to functionally and structurally investigate lipidated GABARAP anchored to nanodiscs, we developed a preparation protocol that is based on chemical linkage of PE to a GABARAP-I variant that has its C-terminal glycine replaced with cysteine. Thus, we modified the expression vector for human GABARAP protein (Swiss-Prot accession number O95166) so as to generate a GABARAP variant containing a C-terminal cysteine that replaces amino acids Gly116 and Leu117 (GABARAP(G116CDL117)). The uniformly N-labeled protein GABARAP(G116CDL117) was expressed and purified as described previously for GABARAP. We covalently linked the thiol-reactive phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (DPPEMBP) to GABARAP(G116CDL117) and assembled nanodiscs containing the lipid-bound protein together with MSP and 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Details of the experimental procedure are given in the Supporting Information. We characterized the structural properties of nanodisc-anchored GABARAP(G116CDL117)-PE by liquid-state NMR spectroscopy. The H,N HSQC spectrum of nanodisc-anchored GABARAP(G116CDL117)-PE is very similar to that of unlipidated wildtype GABARAP (Figure 1). This suggests that the overall fold of the protein is not affected by lipidation and membrane insertion. Resonances in Figure 1 are labeled based on published assignments. Increased line widths of the resonances could be observed for nanodisc-anchored GABARAP(G116CDL117) [a] P. Ma, Dr. J. Mohrl der, M. Schwarten , Dr. M. Stoldt , S. K. Singh, Dr. R. Hartmann, Dr. V. Pacheco , Prof. Dr. D. Willbold Institute of Structural Biology and Biophysics (ISB-3) Research Centre J lich, 52425 J lich (Germany) Fax: (+ 49) 2461-618766 E-mail : j.mohrlueder@fz-juelich.de d.willbold@fz-juelich.de [b] M. Schwarten , Dr. M. Stoldt , Dr. V. Pacheco , Prof. Dr. D. Willbold Institute of Physical Biology and BMFZ, Heinrich-Heine-Universit t D sseldorf 40225 D sseldorf (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000354.