The human Atg8 family member GABARAP is involved in numerous autophagy-related and -unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances epidermal growth factor receptor (EGFR) degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within EGFR, the first of which (LIR1) is selected as a GABARAP binding site in silico. Indeed, in vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable in this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein family's multifaceted roles. We employed live cell imaging with particular emphasis on a GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G-B-G), which retains both a free N-terminus and a lipidation-competent c-terminus, while bivalence creates a gain in affinity conferred by avidity. Notably, reminiscent of early in vitro studies demonstrating an interaction of GABARAP and tubulin, our results revealed a robust association of G-B-G with the microtubule network in living cells. We show that the presence of several basic residues in the amino-terminal helical subdomain of GABARAP and avidity emerged as essential for robust MT association, whereas lipidation ability was not decisive. Interestingly, while the position of the FP-tag had little influence on the result, the nature of the FP itself was crucial, with mTagBFP2 being required for tracking GABARAP tandems in the vicinity of MTs. Though artificial effects cannot be excluded, we assume that G-B-G, with its increased avidity, can give visibility to processes that are based on inherently weak interactions, and thus can help elucidate potential roles of GABARAP e.g. in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.

The human ATG8 family member GABARAP is involved in numerous autophagy-related and - unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances EGFR degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within the EGFR, the first of which (LIR1) is selected as GABARAP binding site in-silico . Indeed, in-vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting a canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding. ### Competing Interest Statement The authors have declared no competing interest. * AF2 : AlphaFold 2 AP2 : adaptor protein complex 2 BLI : biolayer interferometry CASM : conjugation of ATG8 to single membranes CBL : E3 ubiquitin-protein ligase CBL CD36 : platelet glycoprotein 4 CSP : chemical shift perturbation CTD : carboxy-terminal domain DHHC20 : palmitoyltransferase ZDHHC20 DVL2 : segment polarity protein dishevelled homolog DVL-2 EGF : epidermal growth factor EGFR : epidermal growth factor receptor GABAAR : γ-aminobutyric acid type A receptor GABARAP : γ-aminobutyric acid type A receptor-associated protein GRB2 : growth factor receptor-bound protein 2 GST : glutathione S-transferase hATG8 : human autophagy related protein 8 HSQC : heteronuclear single quantum coherence LC3/MAP1LC3 : microtubule-associated protein 1 light chain 3 LDELS : LC3-dependent extracellular vesicle loading and secretion LDS : LIR docking site LIR : LC3-interacting region MAPK/ERK : mitogen-activated protein kinase/extracellular-signal regulated kinases PCM1 : pericentriolar material 1 protein p66SHC : p66 isoform of Src homology 2 domain-containing transforming protein 1 (SHC1) PIK3C3/VPS34 : phosphatidylinositol 3-kinase catalytic subunit type 3/ SCOC : short coiled-coil protein SQSTM1 : sequestosome-1 TLR4 : toll-like receptor 4 TREM2 : triggering receptor expressed on myeloid cells 2 ULK1 : unc-51-like kinase 1

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein family’s multifaceted roles. However, recent findings have unveiled potential limitations of bulky N-terminal tags, particularly regarding ATG8 functionality and localization in specific contexts. This study employed live cell imaging with particular emphasis on the GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G- B -G), which retains both a free N-terminus and a lipidation-competent C-terminus. Notably, our results revealed a robust association of G- B -G with the microtubule network in living cells which was not observed with N-terminal FP fusions of GABARAP, although early in vitro studies demonstrated an interaction of GABARAP and tubulin. Since we observed alteration of the microtubule network organization for G- B -G, this construct emerges as a valuable tool, which can help shedding light on potential roles of GABARAP in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.

The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.

The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.

AbstractBy studying HIV infection, structural biologists are seeking to understand the interactions between the disease‐causing virus and its host. Will this result in new treatments? At the very least, it is shedding new light on fundamental cell mechanisms.

Anhand der HIV‐Infektion wollen Strukturbiologen die Wechselbeziehungen zwischen dem krank machenden Virus und seinem Wirt besser verstehen. Ergeben sich daraus neue therapeutische Ansätze? In jedem Fall Einsichten in grundlegende Zellmechanismen.

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.

triggers local Rac-GTP hydrolysis, thus reducing local actin polymerization required for filopodia formation. ArhGAP44 expression increases as the neuronal network is established and the frequency of exploratory filopodia formation is diminished, suggesting that ArhGAP44 may facilitate the transition of neurons from a dynamic exploratory mode to a mature more static state, a hallmark of nervous system development. Together, our data reveals a local and receptor-independent auto-regulatory mechanism that limits initiation of exploratory filopodia in neurons via protein recruitment to nanoscale membrane deformations.

Dengue virus (DENV) is a mosquito-transmitted virus that causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. There is no vaccine available against DENV and no specific treatment for dengue fever. DENV is believed to replicate its RNA genome in association with modified intracellular membranes. DENV non-structural protein 4A (NS4A) has been implicated in the formation of the viral RNA replication complex (RC). However, the details of RC assembly are incompletely understood. We have previously identified a conserved region in the N-terminal 48 amino acids of NS4A containing putative amphipathic helices (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization [1]. Solution state NMR spectroscopy was used to study the structure of recombinant wild type NS4A a.a. 1-48 peptide and a double mutant NS4A(1-48, L6E;M10E) peptide in the presence of membrane mimicking SDS micelles. The peptides are basically unstructured in aqueous buffer. However, two α-helical segments separated by a non-helical linker are observed for both peptides in presence of SDS micelles. Addition of liposomes induced formation of α-helical secondary structure in the wild type NS4A(1-48) but not in the mutant peptide. We used surface plasmon resonance, floatation assays, and circular dichroism spectroscopy to analyze the binding of recombinant NS4A(1-48) peptides to liposomes. We found that NS4A(1-48) binds to liposomes in a membrane curvature-dependent manner. The AH mutations reduced the affinity of NS4A(1-48) for lipid membranes. These results suggest that the two AHs in the N-terminus of NS4A may be crucial for membrane binding, curvature sensing and stabilization. Better understanding of the molecular details of the DENV RC formation might lead to novel anti-DENV strategies. [1] O. Stern et al. (2013) J. Virol. 87:4080-85

Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1-48), has been proposed to contain an amphipathic α-helix (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization. Nuclear magnetic resonance (NMR) spectroscopy was used to characterize the N-terminal cytoplasmic region (amino acids 1-48) of both wild type and mutant NS4A in the presence of SDS micelles. Binding of the two N-terminal NS4A peptides to liposomes was studied as a function of membrane curvature and lipid composition. The NS4A N-terminal was found to contain two AHs separated by a non-helical linker. The above mentioned mutations did not significantly affect the helical secondary structure of this domain. However, they reduced the affinity of the N-terminal NS4A domain for lipid membranes. Binding of wild type NS4A(1-48) to liposomes is highly dependent on membrane curvature.

Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1-48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1-48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1-48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A.

The mechanism of oxidation of As(III) in alkaline solution by air with promotion effect of KMnO4 was studied. The experimental results indicated that the superstoichiometric oxidation of As(III) by KMnO4 could be attributed to the catalytic effect of reductive product of KMnO4. The XRD and XPS results demonstrated that the catalyst was nascent MnO2 rich in potassium. The results also showed that the mole ratio of Mn/As and the initial pH had significant effects on the oxidation of As(III). The time for the oxidation by air was less than 2 h with the mole ratio of Mn/As less than 1/10.5 and the initial pH higher than 13. The kinetics of the catalytic oxidation of arsenic was interpreted using the pseudo first order reaction, and the apparent active energy was about 15.01 kJ/mol. The study suggested that the initial oxidation was firstly dominated by the direct oxidation of KMnO4 followed by the catalytic oxidation with the nascent MnO2.

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347-361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.

Background Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes. Methodology/Principal Findings This report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded. Conclusions/Significance Small molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.