As chemical inhibitors of autophagy may lead to off-target effects, specific shRNAs of ATG3 and ATG5, which are genes whose protein products are involved in 2 essential ubiquitin-like conjugation systems in autophagy, were used.
We identified 23 putative conjugation systems in the genus, of which 11 were classified as MPFF (family of the F plasmid), 4 MPFI (family of the R64 plasmid), and 8 MPFT (family of the Ti plasmid).
However, cup-shaped membrane isolation and targeting of LC3-II to phagophores needs another conjugation system, in which ATG12 ATG5 conjugates noncovalently associate with ATG16L1 to form a multimeric complex.
Taken together the cellular distribution of LdUfm1, LdUba5, LdUfc1 and the Ufm1 conjugated substrates in the mitochondria argues for a mitochondrion specific Ufm1 conjugation system in Leishmania.
This modification of LC3 is conducted by the ubiquitin-like conjugation system in which Atg7 and Atg3 act as E1 and E2 enzymes, respectively (Tanida et al. 2001).
Formation of the autophagosome involves an ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles.
In addition, MAP1LCB3 and ATG5 are both central in the ubiquitin-like conjugation systems involved in the formation of autophagosomes (Yorimitsu and Klionsky, 2005).
Two conjugation systems involved in the elongation phase, ATG12 and ATG8/LC3, are indispensable for autophagosome formation.
Afterwards, during the later steps of autophagy, two ubiquitin-like protein conjugation systems participate in the elongation and maturation of autophagosome: the Atg12-Atg5-Atg16L complex and the microtubule-associated protein 1 light chain 3 (LC3 -phophatidylethanolamine (PE) complex.
APAP hepatotoxicity is initiated when concentrations of the drug overwhelm the capacity of the conjugation systems, resulting in accumulation of NAPQI, and its formation of protein adducts with cellular proteins, especially in the mitochondria (Jollow et al. 1973; Jaeschke et al. 2012a).
Among the 'core' ATG genes, Atg5 and Atg7 play a significant role in the Atg12-conjugation system and Lc3 (light chain 3), mammalian orthologous gene of Atg8 and encodes MAP1LC3 (microtubule-associated protein 1 light chain 3), is vitally involved in the LC3/Atg8-conjugation system in the elongation step of autophagosome formation process [ 3, 10].
