1-Deoxysphingolipids Tempt Autophagy Resulting in Lysosomal Lipid Substrate Accumulation: Tracing the Impact of 1-Deoxysphingolipids on Ultra-Structural Level using a Novel Click-Chemistry Detection

SUMMARYSphingolipids (SLs) are pivotal components of biological membranes essentially contributing to their physiological functions. 1-deoxysphingolipids (deoxySLs), an atypical cytotoxic acting sub-class of SLs, is relevant for cellular energy homeostasis and is known to be connected to neurodegenerative disorders including diabetic neuropathy and hereditary sensory neuropathy type 1 (HSAN1). High levels of deoxySLs affect lipid membrane integrity in artificial liposomes. Accordingly, recent reports questioned the impact of deoxySLs on physiological lipid membrane and organelle functions leading to impaired cellular energy homeostasis.However, DeoxySL-related structural effects on cell membranes resulting in organelle dysfunction are still obscure. To illuminate disease-relevant sub-cellular targets of deoxySLs, we traced alkyne-containing 1-deoxysphinganine (alkyne-DOXSA) and resulting metabolites on ultra-structural level using a new labeling approach for electron microscopy (EM) termed “Golden-Click-Method” (GCM). To complement high-resolution analysis with membrane dynamics, selected intracellular compartments were traced using fluorescent live dyes.Our results conclusively linked accumulating cytotoxic deoxySLs with mitochondria and endoplasmic reticulum (ER) damage triggering Autophagy of mitochondria and membrane cisterna of the ER. The induced autophagic flux ultimately leads to accumulating deoxySL containing intra-lysosomal lipid crystals. Lysosomal lipid substrate accumulation impaired physiological lysosome functions and caused cellular starvation. Lysosomal exocytosis appeared as a mechanism for cellular clearance of cytotoxic deoxySLs. In sum, our data define new ultra-structural targets of deoxySLs and link membrane damage to autophagy and abnormal lysosomal lipid accumulation. These insights may support new conclusions about diabetes type 2 and HSNA1 related tissue damage.

Transport mechanism of Mycobacterium tuberculosis MmpL/S family proteins and implications in pharmaceutical targeting

Tuberculosis caused by Mycobacterium tuberculosis remains a serious threat to public health. The M. tuberculosis cell envelope is closely related to its virulence and drug resistance. Mycobacterial membrane large proteins (MmpL) are lipid-transporting proteins of the efflux pump resistance nodulation cell division (RND) superfamily with lipid substrate specificity and non-transport lipid function. Mycobacterial membrane small proteins (MmpS) are small regulatory proteins, and they are also responsible for some virulence-related effects as accessory proteins of MmpL. The MmpL transporters are the candidate targets for the development of anti-tuberculosis drugs. This article summarizes the structure, function, phylogenetics of M. tuberculosis MmpL/S proteins and their roles in host immune response, inhibitors and regulatory system.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis

Key Points An AADACL1 ether lipid substrate is phosphorylated in platelets and acts as an endogenous inhibitor of PKC isoforms. AADACL1 inhibition reduces circulating platelet reactivity and modulates thrombosis and hemostasis in vivo.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.