Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes via the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signaling are unknown. We show that Oxysterol Binding Protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA/B, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, mTORC1 recruitment by the Rag GTPases is inhibited due to impaired cholesterol transport to lysosomes. Conversely, OSBPmediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signaling and restores autophagic function in cellular models of NPC. Thus, ER-lysosome contacts are signaling hubs that enable cholesterol sensing by mTORC1, and targeting their sterol-transfer activity could be beneficial in NPC.The exchange of contents and signals between organelles is key to the execution of cellular programs for growth and homeostasis, and failure of this communication can drive disease. A form of organelle communication involves exchange of cholesterol and other lipids by Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Dysregulation of cancer cell metabolism contributes to abnormal cell growth, the biological end point of cancer. We review here numerous affected oncogenes and metabolic pathways common in cancer and how they contribute to cancer pathogenesis and malignancy. This review also discusses various pharmacological manipulations that take advantage of these metabolic abnormalities and the current targeted therapies that have arisen from this research.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is highly abundant in the brain and confers protection against numerous neurological diseases, yet the fundamental mechanisms regulating the enrichment of DHA in the brain remain unknown. Here, we have discovered that a member of the long-chain acyl-CoA synthetase family, Acsl6, is required for the enrichment of DHA in the brain by generating an Acsl6-deficient mouse (Acsl6 −/− ). Acsl6 is highly enriched in the brain and lipid profiling of Acsl6 −/− tissues reveals consistent reductions in DHA-containing lipids in tissues highly abundant with Acsl6. Acsl6 −/− mice demonstrate motor impairments, altered glutamate metabolism, and increased astrogliosis and microglia activation. In response to a neuroinflammatory lipopolysaccharide injection, Acsl6 −/− brains show similar increases in molecular and pathological indices of astrogliosis compared with controls. These data demonstrate that Acsl6 is a key mediator of neuroprotective DHA enrichment in the brain. fatty acid metabolism | neurometabolism | docosahexaenoic acid | acyl-CoA synthetase | brain lipids

Summary All pathogens must acquire nutrients from their hosts. The intracellular bacterial pathogen Legionella pneumophila, the etiological agent of Legionnaires’ disease, requires host amino acids for growth within cells. The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved master regulator of host amino acid metabolism. Here we identify two families of translocated L. pneumophila effector proteins that exhibit opposing effects on mTORC1 activity. The Legionella glucosyltransferase (Lgt) effector family activates mTORC1, through inhibition of host translation, whereas the SidE/SdeABC (SidE) effector family acts as mTORC1 inhibitors. We demonstrate that a common activity of both effector families is to inhibit host translation. We propose that the Lgt and SidE families of effectors work in concert to liberate host amino acids for consumption by L. pneumophila.

Lipid disequilibrium induced by inhibition of long-chain acyl-CoA synthetases impairs ERAD substrate glycan trimming and dislocation independently of its effects on lipid droplet biogenesis. The disruptions in ER proteostasis activate the IRE1 and PERK branches of the unfolded protein response and ultimately induce IRE1-dependent cell death.

A large number of pharmaceuticals, endogenous metabolites, and environmental chemicals act through covalent mechanisms with protein targets. Yet, their specific interactions with the proteome still remain poorly defined for most of these reactive chemicals. Deciphering direct protein targets of reactive small-molecules is critical in understanding their biological action, off-target effects, potential toxicological liabilities, and development of safer and more selective agents. Chemoproteomic technologies have arisen as a powerful strategy that enable the assessment of proteome-wide interactions of these irreversible agents directly in complex biological systems. We review here several chemoproteomic strategies that have facilitated our understanding of specific protein interactions of irreversibly-acting pharmaceuticals, endogenous metabolites, and environmental electrophiles to reveal novel pharmacological, biological, and toxicological mechanisms.

Acetanilide herbicides are among the most widely used pesticides in the United States, but their toxicological potential and mechanisms remain poorly understood. Here, we have used chemoproteomic platforms to map proteome-wide cysteine reactivity of acetochlor (AC), the most widely used acetanilide herbicide, in vivo in mice. We show that AC directly reacts with >20 protein targets in vivo in mouse liver, including the catalytic cysteines of several thiolase enzymes involved in mitochondrial and peroxisomal fatty acid oxidation. We show that the fatty acids that are not oxidized, due to impaired fatty acid oxidation, are instead diverted into other lipid pathways, resulting in heightened free fatty acids, triglycerides, cholesteryl esters, and other lipid species in the liver. Our findings show the utility of chemoproteomic approaches for identifying novel mechanisms of toxicity associated with environmental chemicals like acetanilide herbicides.

Chemical genetics is a powerful approach for identifying therapeutically active small molecules, but identifying the mechanisms of action underlying hit compounds remains challenging. Chemoproteomic platforms have arisen to tackle this challenge and enable rapid mechanistic deconvolution of small-molecule screening hits. Here, we have screened a cysteine-reactive covalent ligand library to identify hit compounds that impair cell survival and proliferation in nonsmall cell lung carcinoma cells, but not in primary human bronchial epithelial cells. Through this screen, we identified a covalent ligand hit, DKM 3-42, which impaired both in situ and in vivo lung cancer pathogenicity. We used activity-based protein profiling to discover that the primary target of DKM 3-42 was the catalytic cysteine in aldehyde dehydrogenase 3A1 (ALDH3A1). We performed further chemoproteomics-enabled covalent ligand screening directly against ALDH3A1, and identified a more potent and selective lead covalent ligand, EN40, which inhibits ALDH3A1 activity and impairs lung cancer pathogenicity. We show here that ALDH3A1 represents a potentially novel therapeutic target for lung cancers that express ALDH3A1 and put forth two selective ALDH3A1 inhibitors. Overall, we show the utility of combining chemical genetics screening of covalent ligand libraries with chemoproteomic approaches to rapidly identify anticancer leads and targets.