Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins.

Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids. To determine whether LC3/GABARAP has other molecular targets, here we generated a pre-processed LC3B mutant (Q116P) that is resistant to ATG4-mediated deconjugation. Upon expression in human cells and when assessed by immunoblotting under reducing and denaturing conditions, deconjugation-resistant LC3B accumulated in multiple forms and at much higher molecular weights than free LC3B. We observed a similar accumulation when pre-processed versions of all mammalian LC3/GABARAP isoforms were expressed in ATG4-deficient cell lines, suggesting that LC3/GABARAP can attach also to other larger molecules. We identified ATG3, the E2-like enzyme involved in LC3/GABARAP lipidation, as one target of conjugation with multiple copies of LC3/GABARAP. We show that LC3B-ATG3 conjugates are distinct from the LC3B-ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B-ATG3 conjugates. Finally, we determined ATG3 residue Lys-243 as an LC3B modification site. Overall, we provide the first cellular evidence that mammalian LC3/GABARAP post-translationally modifies proteins akin to ubiquitination ("LC3ylation"), with ATG4 proteases acting like deubiquitinating enzymes to counteract this modification ("deLC3ylation").

DR WHO: a workshop for house officer preparation.

BACKGROUND: Newly qualified doctors should be competent in advanced life support (ALS) and critical care. The Resuscitation Council has published a course about ALS for undergraduate medical students (the intermediate life support (ILS) course). However, there is no undergraduate-level course on assessing and treating critically ill patients, despite the fact that postgraduate courses on this topic are extremely popular. We have developed a new course called Direct Response Workshop for House Officer Preparation (DR WHO), which teaches both ALS and critical care at an undergraduate level. METHODS: We taught the Resuscitation Council ILS course to our 2003-4 cohort of final year medical students (n = 350), and the new course (DR WHO) to our 2004-5 cohort (n = 338). Students filled in feedback forms immediately after the courses, and a subset repeated the feedback forms 4 months after they had started work as house officers. Course evaluation: Student and house officer feedback was positive. The DR WHO cohort was more confident in caring for critically ill patients (18/26 (69%) were confident after ILS, and 40/45 (89%) were confident after DR WHO (chi2 = 4.3; df = 1; p = 0.06)). Both cohorts were competent in ALS, each with a mean score of 18.6/20 in a finals level practical examination on this topic. CONCLUSIONS: The DR WHO course is popular with the students and practical to run. The course needs to be re-evaluated to determine the long-term competency of graduates.