RESUMO
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.
RESUMO
The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.
Assuntos
Proteínas Associadas aos Microtúbulos , Neoplasias Ovarianas , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Feminino , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeos/farmacologiaRESUMO
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 compositionofTKOs, 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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Membrana Celular/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Transporte ProteicoRESUMO
Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml-1 OD-1 compared with the signal peptide of V. cholerae CtxB (52 mU ml-1 OD-1 ). This positive effect was confirmed with a ß-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher ß-glucosidase activity (181 mU ml-1 OD-1 ) than with the CtxB signal peptide (42 mU ml-1 OD-1 ). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.