2B, right panel). DOPE conjugation to LC3 is shown as comparison (Fig. 3B). The results demonstrate that oxidized PE is an effective substrate for LC3 lipidation, although in this case, it is equally effective as the unoxidized parent lipid. To examine for changes in cellular lipid profiles resulting from 12/15-LOX deficiency, Selleck HSP inhibitor lipidomics profiling of all phospholipid classes and cholesteryl esters was undertaken on lipid extracts from macrophages

obtained from naïve wild type and 12/15-LOX−/− macrophages. There was a tendency overall for increased PE, PI and cholesteryl esters, but decreased PC in 12/15-LOX deficiency. On the other hand, PA, PS and PG were not different. This suggests that loss of the enzyme results OSI-744 molecular weight in a selective defect in particular phospholipid classes at the expense of others (Fig. 3). Herein, we show that deficiency of the lipid-oxidizing enzyme, 12/15-LOX, is associated with altered cellular membrane structure. We also demonstrate that

a LOX-derived oxidized phospholipid is an effective substrate for lipidation of both LC3 and Atg8, being preferred over the unoxidized analog in the case of the yeast homolog. This is suggestive of this pathway being involved in regulation of membrane dynamics. Last, we show altered phospholipid content in murine macrophages deficient in 12/15-LOX. Our observations of double membrane structures suggestive of autophagosomes propose a role in autophagy. Normal LC3 expression and lipidation indicate that the defect in the 12/15-LOX−/− macrophages is likely to be upstream of LC3 activity

itself. 12/15-LOX was first described as the human homolog, 15-LOX1, as being highly induced in bleeding anemia in rabbits, inducing significant peroxidation of intracellular membranes that coincided with disappearance of organelles [19], [20], [21], [22] and [23]. Thus, it was proposed as being critically required for reticulocyte maturation into erythrocytes. However subsequent to this, mice deficient in the functional homolog, 12/15-LOX were shown to have normal red cell counts, and interest in this pathway waned [24]. This does not exclude Metalloexopeptidase that the knockout mice have developed a compensatory mechanism, and that the enzyme still plays a role in normal turnover of organelles during homeostasis. In support of a role for LOX in processes that involve membrane remodeling, previous studies have shown that 12/15-LOX−/− macrophages are unable to undergo a full phagocytosis response towards apoptotic thymocytes [25]. The multiple differences between wild type and 12/15-LOX−/− macrophages seen, including abnormal mitochondria, multiple lysosomal storage bodies and suspected autophagosomes are consistent with LSDs [26], [27], [28] and [29]. Lysosomes are small vesicular organelles, their primary function being to merge with late endosomes to digest their content [30], [31] and [32]. Endosomal degradation is carried out by numerous lipid and protein hydrolases.