OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation

The GAP activity of OATL1, which is recruited to autophagosomes by Atg8, regulates autophagosome–lysosome fusion

Identifizierung von physiologischen und artifiziellen Liganden von GABARAP und Charakterisierung der resultierenden Interaktionen

$\gamma$-aminobutyric acid (GABA) receptors (type A) (GABA$_{A}$ receptors) mediate fast synaptic inhibition in the central nervous system. They are of particular pharmacological importance and are targets for drugs used to treat mental disorders or to modulate sleep and mood. Sorting, transport and degradation of neurotransmitter receptors are important for the construction and maintenance of functional synapses and are fundamental processes to modulate synaptic plasticity. The GABA$_{A}$ receptor-associated protein (GABARAP) belongs to the MAP-LC3 protein family which is involved in vesicular transport processes, like autophagocytosis and intra-golgi transport. GABARAP binds to a GABA$_{A}$ receptor subunit and participates in its transport events. The exact mechanisms and the function of this interaction are not yet known. The objective of the present work was the i dentificati on of artificial peptide and physiological protein ligands of GABARAP and the characterisation of the resulting interactions. This enhances on the one hand the understanding of the binding mechanisms of any ligand to GABARAP and on the other hand, can facilitate the design of new drugs capable of modulating the function of GABARAP or the GABA$_{A}$ receptor. The knowledge of new GABARAP interaction partners additionally contributes to a better understanding of the cellular functions of GABARAP and the procedures at the postsynaptic membrane. By employing a phage display selection procedure, artificial high-affinity peptide ligands of GABARAP could successfully be identified. The resulting interactions could be characterised via enzyme-linked immunosorbent assay, fluorescence titration, surface-plasmon resonance and nuclear magnetic resonance spectroscopy. A consensus motif was derived from the GABARAP-binding peptide sequences. It was used to search protein databases to identify putative GABARAP-binding proteins. For the first time, the human cellular proteins calreticulin (CRT) and clathrin heavy chain (CHC) were identified to bind GABARAP. The corresponding interactions could be characterised via surface plasmon resonance, pulldown analysis and nuclear magnetic resonance spectroscopy. The results of the present work make a substantial contribution to an enhanced understanding of the GABARAP binding specificity and its cellular function

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

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) 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 Ca(2+) -dependent chaperone and a Ca(2+) -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 K(d) = 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

Toto Lotto árusítóhely

MohrlüderA Lotto szó kezdőbetűjével összeolvadó, lóherés, piros kalapot viselő lottófigura kacsint ránk a képről és jobb kezének hüvelykujját felfelé fordítva biztat bennünket vásárlásra

Structural characterization of GABARAP-ligand interactions

The GABA(A) receptor-associated protein (GABARAP) plays an important role in intracellular trafficking of several proteins. It undergoes a C-terminal lipidation process that enables anchoring in the cytosolic leaflet of cellular membranes. While the three-dimensional structure of GABARAP itself has been determined, structural investigation of complexes with its interaction partners has just commenced. Studies with indole derivatives revealed that GABARAP features two hydrophobic binding sites (hp1 and hp2). These also play an essential role in complex formation with the native ligand calreticulin. Furthermore, a model of hexameric N-ethylmaleimide-sensitive factor (NSF) suggests that binding of GABARAP to this molecular machine may involve a similar site. Since hp1 and hp2 are highly conserved throughout the GABARAP family, the relevance of the structural data presented here is likely to extend to GABARAP homologues

Inhibition of Polyglutamine Misfolding with D-Enantiomeric Peptides Identified by Mirror Image Phage Display Selection

Nine heritable diseases are known that are caused by unphysiologically elongated polyglutamine tracts in human proteins leading to misfolding, aggregation and neurodegeneration. Current therapeutic strategies include efforts to inhibit the expression of the respective gene coding for the polyglutamine-containing proteins. There are, however, concerns that this may interfere with the physiological function of the respective protein. We aim to stabilize the protein’s native conformation by D-enantiomeric peptide ligands to prevent misfolding and aggregation, shift the equilibrium between aggregates and monomers towards monomers and dissolve already existing aggregates into non-toxic and functional monomers. Here, we performed a mirror image phage display selection on the polyglutamine containing a fragment of the androgen receptor. An elongated polyglutamine tract in the androgen receptor causes spinal and bulbar muscular atrophy (SBMA). The selected D-enantiomeric peptides were tested for their ability to inhibit polyglutamine-induced androgen receptor aggregation. We identified D-enantiomeric peptide QF2D-2 (sqsqwstpqGkwshwprrr) as the most promising candidate. It binds to an androgen receptor fragment with 46 consecutive glutamine residues and decelerates its aggregation, even in seeded experiments. Therefore, QF2D-2 may be a promising drug candidate for SBMA treatment or even for all nine heritable polyglutamine diseases, since its aggregation-inhibiting property was shown also for a more general polyglutamine target

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