Exquisite exposure: Formaldehyde as a metabolic regulator.

Throughout life, whether through external consumption or internal production, we are exposed to different reactive metabolites considered toxic to the body. Pham et al.1 uncover metabolic regulation by one such harmful metabolite: formaldehyde.

Principles and functions of metabolic compartmentalization.

Metabolism has historically been studied at the levels of whole cells, whole tissues and whole organisms. As a result, our understanding of how compartmentalization-the spatial and temporal separation of pathways and components-shapes organismal metabolism remains limited. At its essence, metabolic compartmentalization fulfils three important functions or 'pillars': establishing unique chemical environments, providing protection from reactive metabolites and enabling the regulation of metabolic pathways. However, how these pillars are established, regulated and maintained at both the cellular and systemic levels remains unclear. Here we discuss how the three pillars are established, maintained and regulated within the cell and discuss the consequences of dysregulation of metabolic compartmentalization in human disease. Organelles are increasingly emerging as 'command-and-control centres' and the increased understanding of metabolic compartmentalization is revealing new aspects of metabolic homeostasis, with this knowledge being translated into therapies for the treatment of cancer and certain neurodegenerative diseases.

MCART1/SLC25A51 is required for mitochondrial NAD transport.

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as coessential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

Mitochondrial metabolism promotes adaptation to proteotoxic stress.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

PNPLA3, CGI-58, and Inhibition of Hepatic Triglyceride Hydrolysis in Mice.

A variant (148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a major risk factor for fatty liver disease. Despite its clinical importance, the pathogenic mechanism linking the variant to liver disease remains poorly defined. Previously, we showed that PNPLA3(148M) accumulates to high levels on hepatic lipid droplets (LDs). Here we examined the effect of that accumulation on triglyceride (TG) hydrolysis by adipose triglyceride lipase (ATGL), the major lipase in the liver. As expected, overexpression of ATGL in cultured hepatoma (HuH-7) cells depleted the cells of LDs, but unexpectedly, co-expression of PNPLA3(wild type [WT] or 148M) with ATGL inhibited that depletion. The inhibitory effect of PNPLA3 was not caused by the displacement of ATGL from LDs. We tested the hypothesis that PNPLA3 interferes with ATGL activity by interacting with its cofactor, comparative gene identification-58 (CGI-58). Evidence supporting such an interaction came from two findings. First, co-expression of PNPLA3 and CGI-58 resulted in LD depletion in cultured cells, but expression of PNPLA3 alone did not. Second, PNPLA3 failed to localize to hepatic LDs in liver-specific Cgi-58 knockout (KO) mice. Moreover, overexpression of PNPLA3(148M) increased hepatic TG levels in WT, but not in Cgi-58 KO mice. Thus, the pro-steatotic effects of PNPLA3 required the presence of CGI-58. Co-immunoprecipitation and pulldown experiments in livers of mice and in vitro using purified proteins provided evidence that PNPLA3 and CGI-58 can interact directly. Conclusion: Taken together, these findings are consistent with a model in which PNPLA3(148M) promotes steatosis by CGI-58-dependent inhibition of ATGL on LDs.

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase.

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.

Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism.

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.