Peixiang Ma

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.

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.

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.

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the proteins interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

A magnetic nanoparticles relaxation sensor for protein–protein interaction detection at ultra-low magnetic field

Functionalized magnetic nanoparticles (MNPs) can serve as magnetic relaxation sensors (MRSs) to detect different biological targets, because the clustering of magnetic particle may cause the spin-spin relaxation time (T2) decrease of the surrounding water protons. However, the application of MNPs in clinical NMR systems faces the challenge of poor stability at magnetic field strengths in the order of tesla. The recently developed ultra-low field (ULF) NMR technique working at microtesla (μT) range then becomes a candidate. Herein, we incorporated superconducting quantum interference device (SQUID) as the detector in the ultra-low field system to enhance the sensitivity. We functionalized the Fe3O4 nanoparticles with the gama-aminobutyrate type A receptor-associated proteins (GABARAP), which specifically interact with calreticulin (CRT). As a result of the interaction between GABARAP and CRT, the clustering of the functionalized MNPs generates local magnetic fields, which accelerate the dephasing of the water protons in the vicinity. We analyzed the relation between T2 values and the CRT concentrations at 211μT and the low detection limit for CRT is 10 pg/ml, which is superior to the immunoblot system. The high sensitivity of the ULF NMR system for protein-protein interaction detection demonstrates the potential to use this inexpensive, portable system for quick biochemical and clinical assays.

Assessment of GABARAP self-association by its diffusion properties

Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) belongs to a family of small ubiquitin-like adaptor proteins implicated in intracellular vesicle trafficking and autophagy. We have used diffusion-ordered nuclear magnetic resonance spectroscopy to study the temperature and concentration dependence of the diffusion properties of GABARAP. Our data suggest the presence of distinct conformational states and provide support for self-association of GABARAP molecules. Assuming a monomer–dimer equilibrium, a temperature-dependent dissociation constant could be derived. Based on a temperature series of 1H15N heteronuclear single quantum coherence nuclear magnetic resonance spectra, we propose residues potentially involved in GABARAP self-interaction. The possible biological significance of these observations is discussed with respect to alternative scenarios of oligomerization.

Conformational Polymorphism in Autophagy-Related Protein GATE-16

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.