GABARAPL1 antibodies: Target one protein,get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA_A receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

GABARAPL1 (GEC1): original or copycat?

The GABARAPL1 (GABARAP-LIKE 1) gene was first described as an early estrogen-regulated gene that shares a high sequence homology with GABARAP and is thus a part of the GABARAP family. GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. The GABARAP family members (GABARAP, GABARAPL1 and GABARAPL2) and their close homologs (LC3 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. However, despite these similarities, GABARAPL1 displays a complex regulation that is different from that of other GABARAP family members. Moreover, it presents a regulated tissue expression and is the most highly expressed gene among the family in the central nervous system. In this review article, we will outline the specific functions of this protein and also hypothesize about the roles that GABARAPL1 might have in several important biological processes such as cancer or neurodegenerative diseases.     

GABARAPL1 (GEC1): Original or copycat?

The GABARAPL1 (GABARAP-LIKE 1) gene was first described as an early estrogen-regulated gene that shares a high sequence homology with GABARAP and is thus a part of the GABARAP family. GABARAPL1, like GABARAP, interacts with the GABA_A receptor and tubulin and promotes tubulin polymerization. The GABARAP family members (GABARAP, GABARAPL1 and GABARAPL2) and their close homologs (LC3 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. However, despite these similarities, GABARAPL1 displays a complex regulation that is different from that of other GABARAP family members. Moreover, it presents a regulated tissue expression and is the most highly expressed gene among the family in the central nervous system. In this review article, we will outline the specific functions of this protein and also hypothesize about the roles that GABARAPL1 might have in several important biological processes such as cancer or neurodegenerative diseases.     

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.     

GABARAPL1 (GEC1) associates with autophagic vesicles

Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.     

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.     

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B,GABARAPL2 and GABARAP

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.     

An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5

ABSTRACT

Short linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.

Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5     

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

人类GABARAPL2基因的亚细胞定位

为了对GABARAPL2（GABAA受体相关蛋白相似蛋白2）基因的功能进行初步分析,首先通过同源比较的方法将序列与其同源物进行比较,发现GABARAPL2的氨基酸序列与GABARAP（GABAA受体相关蛋白）高度同源,而GABARAP已证实通过结合细胞骨架的微蛋白,使GABAA受体聚集,定位在细胞膜上,本文采用PCR法从人脑组织的cDNA文库中扩增出GABARAPL2的cDNA,克隆至T质粒载体中进行测序验证,然后以此为模板引物中引入酶切位点再次PCR,扩增出GABARAPL2的开放阅读框,并将其插入到加强型绿色荧光蛋白融合表达载体EGFP中,将绿色荧光蛋白标记的GABARAPL2和GABARAP分别转染HLF细胞株,结果两种蛋白的分布情况基本一致,在细胞质内和核内均有分布,而且核内的分布较胞质为多,结构功能域分析表明,GABARAPL2含有蛋白激酶C磷酸化位点和酪氨酸激酶磷酸化位点,可能通过磷酸化参与细胞骨架的变化,结论 GABARAPL2和GABARAP不仅在胞质中作为受体相关蛋白协助受体的聚集、定位,还参与体内许多其它重要的生理过程。     

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.     

Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones.

The subfamily of guanine nucleotide-binding regulatory (G proteins) designated Gq has been shown to regulate the activity of phospholipase C by reconstitution. However, the role of these proteins in hormonal regulation of this activity has not been demonstrated. Two antisera were used in attempts to interrupt this pathway. Antiserum W082, developed against a peptide representing an internal sequence in alpha q, was specific for alpha q by immunoblots but did not recognize the native protein. Antiserum X384 was developed against a peptide representing the 12 amino acids of the common carboxyl termini of alpha q and alpha 11. It had a broader specificity for this subfamily of G protein alpha subunits and recognized the native proteins. Antiserum X384 specifically immunoprecipitated alpha q and its homologs from purified preparations and detergent extracts of membranes. Affinity-purified antibodies attenuated stimulation of phosphatidylinositide 4,5-bisphosphate hydrolysis by bradykinin, angiotensin, and histamine in membranes derived from NG108-15 cells, rat liver, and 1321N1 cells, respectively. Activation of the phospholipase C activity by guanosine 5'-3-O-(thio)triphosphate alone was also inhibited. Inclusion of the peptide to which the antisera were raised blocked the effect of the antibody. In contrast, affinity-purified W082, which did not recognize native proteins, did not alter regulation of phospholipase C. This indicates that the Gq family of signaling proteins can couple to several receptors and is responsible for the hormonal regulation of phospholipase C in these diverse systems. The further generality of this regulatory pathway remains to be established.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

Phosphatidylinositol 4‐phosphate on Rab7‐positive autophagosomes revealed by the freeze‐fracture replica labeling

Phosphatidylinositol 4‐phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P‐binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP‐binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze‐fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome‐lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.