Publications by authors named "Alan Saghatelian"

139 Publications

Distinct Biological Activities of Isomers from Different Families of Branched Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs).

J Lipid Res 2021 Aug 18:100108. Epub 2021 Aug 18.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215. Electronic address:

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. Each FAHFA family consists of esters with different acyl chains and multiple isomers with branchpoints at different carbons. Some FAHFAs, including palmitic acid hydroxy stearic acids (PAHSAs), have been shown to improve insulin sensitivity and glucose tolerance in mice by enhancing glucose-stimulated insulin secretion (GSIS), insulin-stimulated glucose transport, insulin action to suppress hepatic glucose production and reducing adipose tissue inflammation. However, little is known about the biological effects of other FAHFAs. Here, we investigated whether PAHSAs, oleic, pamitoleic, and stearic acid hydroxy stearic acids (OAHSAs, POHSAs, and SAHSAs) potentiate GSIS in β-cells and human islets, insulin-stimulated glucose uptake in adipocytes, and anti-inflammatory effects in immune cells. We also investigated whether they activate G protein couple receptor (GPR40), which has been shown to mediate the effects of PAHSAs on insulin secretion and sensitivity in vivo. We show that many FAHFAs potentiate GSIS, activate GPR40, and attenuate LPS-induced chemokine and cytokine expression and secretion and phagocytosis in immune cells. However, fewer FAHFAs augment insulin-stimulated glucose uptake in adipocytes. S-9-PAHSA, but not R-9-PAHSA, potentiated GSIS and glucose uptake, while both stereoisomers had anti-inflammatory effects. The differential effects on GSIS do not result from differences in GPR40 activation. FAHFAs containing unsaturated acyl chains with higher branching from the carboxylate head group are more likely to potentiate GSIS while FAHFAs with lower branching are more likely to be anti-inflammatory. Thus, different FAHFA isomers have distinct physiological functions.
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http://dx.doi.org/10.1016/j.jlr.2021.100108DOI Listing
August 2021

Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition.

J Cell Biol 2021 Aug 16;220(8). Epub 2021 Jun 16.

Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, Irvine, CA.

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.
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http://dx.doi.org/10.1083/jcb.202101063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240855PMC
August 2021

A short ORF-encoded transcriptional regulator.

Proc Natl Acad Sci U S A 2021 01;118(4)

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
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http://dx.doi.org/10.1073/pnas.2021943118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848545PMC
January 2021

Elovl2 Is Required for Robust Visual Function in Zebrafish.

Cells 2020 12 2;9(12). Epub 2020 Dec 2.

Viterbi Family Department of Ophthalmology, Shiley Eye Institute, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) play critical roles in membrane stability and cell signaling within the retina. ELOVL2 (Elongation of Very Long Chain Fatty Acids-Like 2), an elongase involved in the synthesis of long chain polyunsaturated fatty acids (LC-PUFAs), has recently been implicated in regulating aging in the mammalian retina. In this work, we characterize the expression and function of in the retina development in embryonic zebrafish. Whole mount in situ hybridization shows is expressed in the Muller glia in embryonic and adult zebrafish. Lipidomics analysis of crispants whole embryos at day 2 and eyes at day 7 demonstrated significant changes in lipids composition, especially on the level of lipids containing docosahexaenoic acid (DHA). Histological analysis of zebrafish lacking revealed increased retinal thickness compared to controls at day 7 without gross disruptions of the retinal architecture. Finally, crispants showed differences in the visual motor reflex light off (VMR-OFF) at day 7 compared to controls. In sum, inactivation of in zebrafish embryos caused changes in lipid composition and in visual behavior, further confirming the important role of LC-PUFAs in healthy vision.
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http://dx.doi.org/10.3390/cells9122583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761535PMC
December 2020

Networking in Circulation: Lipoproteins, PM20D1, and N-acyl Amino Acid Bioactivity.

Authors:
Alan Saghatelian

Cell Chem Biol 2020 09;27(9):1112-1113

The Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA. Electronic address:

N-acyl amino acids are a class of biologically active lipids that control thermogenesis, and their biosynthesis is facilitated by PM20D1. In this issue of Cell Chemical Biology, Kim et al. (2020) identify a lipoprotein-albumin network in the blood that regulates physiological levels of N-acyl amino acids.
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http://dx.doi.org/10.1016/j.chembiol.2020.08.019DOI Listing
September 2020

FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis.

J Cell Biol 2020 10;219(10)

Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA.

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis.
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http://dx.doi.org/10.1083/jcb.202006111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659722PMC
October 2020

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Nature 2020 10 12;586(7831):790-795. Epub 2020 Aug 12.

Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC2-or in conditions of low serine availability-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.
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http://dx.doi.org/10.1038/s41586-020-2609-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606299PMC
October 2020

Directed remodeling of the mouse gut microbiome inhibits the development of atherosclerosis.

Nat Biotechnol 2020 11 15;38(11):1288-1297. Epub 2020 Jun 15.

Department of Chemistry, Scripps Research, La Jolla, CA, USA.

The gut microbiome is a malleable microbial community that can remodel in response to various factors, including diet, and contribute to the development of several chronic diseases, including atherosclerosis. We devised an in vitro screening protocol of the mouse gut microbiome to discover molecules that can selectively modify bacterial growth. This approach was used to identify cyclic D,L-α-peptides that remodeled the Western diet (WD) gut microbiome toward the low-fat-diet microbiome state. Daily oral administration of the peptides in WD-fed LDLr mice reduced plasma total cholesterol levels and atherosclerotic plaques. Depletion of the microbiome with antibiotics abrogated these effects. Peptide treatment reprogrammed the microbiome transcriptome, suppressed the production of pro-inflammatory cytokines (including interleukin-6, tumor necrosis factor-α and interleukin-1β), rebalanced levels of short-chain fatty acids and bile acids, improved gut barrier integrity and increased intestinal T regulatory cells. Directed chemical manipulation provides an additional tool for deciphering the chemical biology of the gut microbiome and might advance microbiome-targeted therapeutics.
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http://dx.doi.org/10.1038/s41587-020-0549-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641989PMC
November 2020

Antioxidant Effects of N-Acetylcysteine Prevent Programmed Metabolic Disease in Mice.

Diabetes 2020 08 22;69(8):1650-1661. Epub 2020 May 22.

Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, New York, NY.

An adverse maternal in utero and lactation environment can program offspring for increased risk for metabolic disease. The aim of this study was to determine whether N-acetylcysteine (NAC), an anti-inflammatory antioxidant, attenuates programmed susceptibility to obesity and insulin resistance in offspring of mothers on a high-fat diet (HFD) during pregnancy. CD1 female mice were acutely fed a standard breeding chow or HFD. NAC was added to the drinking water (1 g/kg) of the treatment cohorts from embryonic day 0.5 until the end of lactation. NAC treatment normalized HFD-induced maternal weight gain and oxidative stress, improved the maternal lipidome, and prevented maternal leptin resistance. These favorable changes in the in utero environment normalized postnatal growth, decreased white adipose tissue (WAT) and hepatic fat, improved glucose and insulin tolerance and antioxidant capacity, reduced leptin and insulin, and increased adiponectin in HFD offspring. The lifelong metabolic improvements in the offspring were accompanied by reductions in proinflammatory gene expression in liver and WAT and increased thermogenic gene expression in brown adipose tissue. These results, for the first time, provide a mechanistic rationale for how NAC can prevent the onset of metabolic disease in the offspring of mothers who consume a typical Western HFD.
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http://dx.doi.org/10.2337/db19-1129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372077PMC
August 2020

Insights into GLP-1 Receptor Activation with a Nonpeptide Agonist.

Biochemistry 2020 04 16;59(16):1549-1550. Epub 2020 Apr 16.

Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037-1002, United States.

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http://dx.doi.org/10.1021/acs.biochem.0c00180DOI Listing
April 2020

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice.

J Biol Chem 2020 05 9;295(18):5891-5905. Epub 2020 Mar 9.

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037. Electronic address:

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9 carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.
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http://dx.doi.org/10.1074/jbc.RA119.012145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196635PMC
May 2020

The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.

Aging Cell 2020 02 14;19(2):e13100. Epub 2020 Jan 14.

Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA.

Methylation of the regulatory region of the elongation of very-long-chain fatty acids-like 2 (ELOVL2) gene, an enzyme involved in elongation of long-chain polyunsaturated fatty acids, is one of the most robust biomarkers of human age, but the critical question of whether ELOVL2 plays a functional role in molecular aging has not been resolved. Here, we report that Elovl2 regulates age-associated functional and anatomical aging in vivo, focusing on mouse retina, with direct relevance to age-related eye diseases. We show that an age-related decrease in Elovl2 expression is associated with increased DNA methylation of its promoter. Reversal of Elovl2 promoter hypermethylation in vivo through intravitreal injection of 5-Aza-2'-deoxycytidine (5-Aza-dc) leads to increased Elovl2 expression and rescue of age-related decline in visual function. Mice carrying a point mutation C234W that disrupts Elovl2-specific enzymatic activity show electrophysiological characteristics of premature visual decline, as well as early appearance of autofluorescent deposits, well-established markers of aging in the mouse retina. Finally, we find deposits underneath the retinal pigment epithelium in Elovl2 mutant mice, containing components found in human drusen, a pathologic hallmark of age related macular degeneration. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual function, and suggest novel therapeutic strategies for the treatment of age-related eye diseases.
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http://dx.doi.org/10.1111/acel.13100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996962PMC
February 2020

Accurate annotation of human protein-coding small open reading frames.

Nat Chem Biol 2020 04 9;16(4):458-468. Epub 2019 Dec 9.

Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, CA, USA.

Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs. Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes.
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http://dx.doi.org/10.1038/s41589-019-0425-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085969PMC
April 2020

Regulation of the ER stress response by a mitochondrial microprotein.

Nat Commun 2019 10 25;10(1):4883. Epub 2019 Oct 25.

The Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010N. Torrey Pines Rd, La Jolla, CA, 92037, USA.

Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER-mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.
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http://dx.doi.org/10.1038/s41467-019-12816-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814811PMC
October 2019

Stereochemistry of Linoleic Acid Esters of Hydroxy Linoleic Acids.

Org Lett 2019 10 23;21(19):8080-8084. Epub 2019 Sep 23.

Skaggs School of Pharmacy and Pharmaceutical Sciences , UC San Diego , 9500 Gilman Drive , La Jolla , California 92093-0934 , United States.

The syntheses of linoleic acid esters of hydroxy linoleic acids (LAHLAs) present in oat oil and human serum have been achieved, providing access to material for testing and the determination of the stereochemistry of the natural compounds. While 9- and 13-LAHLAs were found to be a mixture of enantiomers 15-LAHLA is generated in a single optical form in oat oil. The stereochemistry of 15-LAHLA in oat oil was found to be opposite to that reported for digalactosyldiacylglycerol that possesses an embedded 15-LAHLA.
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http://dx.doi.org/10.1021/acs.orglett.9b03054DOI Listing
October 2019

PAHSAs enhance hepatic and systemic insulin sensitivity through direct and indirect mechanisms.

J Clin Invest 2019 10;129(10):4138-4150

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet-fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs' effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein-coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.
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http://dx.doi.org/10.1172/JCI127092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763232PMC
October 2019

Substrate-Selective Enzyme Inhibitors.

Trends Pharmacol Sci 2019 10 2;40(10):716-718. Epub 2019 Aug 2.

The Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA. Electronic address:

Enzymes with multiple substrates pose a unique challenge for drug development because of an increased potential for on-target side effects. Maianti and colleagues (Nat. Chem. Biol., 2019) identify novel exo-site inhibitors with abilities to alter the substrate-selectivity of insulin-degrading enzymes (IDE). Their work illuminates new therapeutic avenues for discovering small-molecule enzyme inhibitors and redefines our current understanding of drugging enzymes with multiple substrates.
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http://dx.doi.org/10.1016/j.tips.2019.07.009DOI Listing
October 2019

PAHSAs attenuate immune responses and promote β cell survival in autoimmune diabetic mice.

J Clin Invest 2019 08 5;129(9):3717-3731. Epub 2019 Aug 5.

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.

Palmitic acid esters of hydroxy stearic acids (PAHSAs) are endogenous antidiabetic and antiinflammatory lipids. Here, we show that PAHSAs protect against type 1 diabetes (T1D) and promote β cell survival and function. Daily oral PAHSA administration to nonobese diabetic (NOD) mice delayed the onset of T1D and markedly reduced the incidence of T1D, whether PAHSAs were started before or after insulitis was established. PAHSAs reduced T and B cell infiltration and CD4+ and CD8+ T cell activation, while increasing Treg activation in pancreata of NOD mice. PAHSAs promoted β cell proliferation in both NOD mice and MIN6 cells and increased the number of β cells in NOD mice. PAHSAs attenuated cytokine-induced apoptotic and necrotic β cell death and increased β cell viability. The mechanism appears to involve a reduction of ER stress and MAPK signaling, since PAHSAs lowered ER stress in NOD mice, suppressed thapsigargin-induced PARP cleavage in human islets, and attenuated ERK1/2 and JNK1/2 activation in MIN6 cells. This appeared to be mediated in part by glucagon-like peptide 1 receptor (GLP-1R) and not the G protein-coupled receptor GPR40. PAHSAs also prevented impairment of glucose-stimulated insulin secretion and improved glucose tolerance in NOD mice. Thus, PAHSAs delayed the onset of T1D and reduced its incidence by attenuating immune responses and exerting direct protective effects on β cell survival and function.
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http://dx.doi.org/10.1172/JCI122445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715391PMC
August 2019

Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and mammals.

J Biol Chem 2019 07 31;294(27):10698-10707. Epub 2019 May 31.

From the Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California 92037,

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.
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http://dx.doi.org/10.1074/jbc.RA118.006956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615670PMC
July 2019

Discovery of FAHFA-Containing Triacylglycerols and Their Metabolic Regulation.

J Am Chem Soc 2019 06 13;141(22):8798-8806. Epub 2019 May 13.

Clayton Foundation Laboratories for Peptide Biology , Salk Institute for Biological Studies , 10010 North Torrey Pines Road , La Jolla , California 92037-1002 , United States.

FAHFAs are a class of bioactive lipids, which show great promise for treating diabetes and inflammatory diseases. Deciphering the metabolic pathways that regulate endogenous FAHFA levels is critical for developing diagnostic and therapeutic strategies. However, it remains unclear how FAHFAs are metabolized in cells or tissues. Here, we investigate whether FAHFAs can be incorporated into other lipid classes and identify a novel class of endogenous lipids, FAHFA-containing triacylglycerols (FAHFA-TGs), which contain a FAHFA group esterified to the glycerol backbone. Isotope-labeled FAHFAs are incorporated into FAHFA-TGs when added to differentiated adipocytes, which implies the existence of enzymes and metabolic pathways capable of synthesizing these lipids. Induction of lipolysis (i.e., triacylglycerol hydrolysis) in adipocytes is associated with marked increases in nonesterified FAHFA levels, demonstrating that FAHFA-TGs breakdown is a regulator of cellular FAHFA levels. To quantify FAHFA levels in FAHFA-TGs and determine their regioisomeric distributions, we developed a mild alkaline hydrolysis method that liberates FAHFAs from triacylglycerols for easier detection. FAHFA-TG concentrations are greater than 100-fold than that of nonesterified FAHFAs, indicating that FAHFA-TGs are a major reservoir of FAHFAs in cells and tissues. The discovery of FAHFA-TGs reveals a new branch of TG and FAHFA metabolism with potential roles in metabolic health and regulation of inflammation.
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http://dx.doi.org/10.1021/jacs.9b00045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662584PMC
June 2019

Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA.

J Antibiot (Tokyo) 2019 06 15;72(6):498-506. Epub 2019 Apr 15.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0934, USA.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a growing class of natural products found in organisms ranging from plants to humans. The roles these endogenous derivatives of fatty acids play in biology and their novel pathways for controlling inflammation have increased our understanding of basic human physiology. FAHFAs incorporate diverse fatty acids into their structures, however, given their recent discovery non-natural derivatives have not been a focus and as a result structure-activity relationships remain unknown. The importance of the long chain hydrocarbons extending from the ester linkage as they relate to anti-inflammatory activity is unknown. Herein the systematic removal of carbons from either the hydroxy fatty acid or fatty acid regions of the most studied FAHFA, palmitic acid ester of 9-hydroxystearic acid (9-PAHSA), was achieved and these synthetic, abridged analogs were tested for their ability to attenuate IL-6 production. Reduction of the carbon chain lengths of the 9-hydroxystearic acid portion or palmitic acid hydrocarbon chain resulted in lower molecular weight analogs that maintained anti-inflammatory activity or in one case enhanced activity.
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http://dx.doi.org/10.1038/s41429-019-0180-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784544PMC
June 2019

CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability.

Genes Dev 2019 04 28;33(7-8):418-435. Epub 2019 Feb 28.

Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.
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http://dx.doi.org/10.1101/gad.322339.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446539PMC
April 2019

A Hidden ORF Reveals an Immune Protector.

Biochemistry 2019 02 6;58(8):1022-1023. Epub 2019 Feb 6.

Clayton Foundation Laboratories for Peptide Biology , Salk Institute for Biological Studies , 10010 North Torrey Pines Road , La Jolla , California 92037-1002 , United States.

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http://dx.doi.org/10.1021/acs.biochem.8b01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443401PMC
February 2019

Genetic Liver-Specific AMPK Activation Protects against Diet-Induced Obesity and NAFLD.

Cell Rep 2019 01;26(1):192-208.e6

Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address:

The AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD, characterized by excessive accumulation of hepatic lipids, is the most common chronic liver disease and a major risk factor for development of nonalcoholic steatohepatitis, type 2 diabetes, and other metabolic conditions. To assess the therapeutic potential of AMPK activation, we have generated a genetically engineered mouse model, termed iAMPK, where AMPK can be inducibly activated in vivo in mice in a spatially and temporally restricted manner. Using this model, we show that liver-specific AMPK activation reprograms lipid metabolism, reduces liver steatosis, decreases expression of inflammation and fibrosis genes, and leads to significant therapeutic benefits in the context of diet-induced obesity. These findings further support AMPK as a target for the prevention and treatment of NAFLD.
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http://dx.doi.org/10.1016/j.celrep.2018.12.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344045PMC
January 2019

Small, but mighty? Searching for human microproteins and their potential for understanding health and disease.

Expert Rev Proteomics 2018 12 15;15(12):963-965. Epub 2018 Nov 15.

a Clayton Foundation Laboratories for Peptide Biology , The Salk Institute for Biological Studies , La Jolla , CA , USA.

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http://dx.doi.org/10.1080/14789450.2018.1547194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497149PMC
December 2018

Methodological Issues in Studying PAHSA Biology: Masking PAHSA Effects.

Cell Metab 2018 10 20;28(4):543-546. Epub 2018 Sep 20.

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

PAHSAs are anti-diabetic and anti-inflammatory lipids. Syed et al. identify numerous experimental differences that likely account for the failure of Pflimlin et al. to observe PAHSA beneficial effects. The differences include different HFDs resulting in minimal/no glucose intolerance, different assay conditions, an LC-MS protocol that was not validated, and use of olive oil, a bioactive nutrient that improves glucose tolerance, as a vehicle.
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http://dx.doi.org/10.1016/j.cmet.2018.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542592PMC
October 2018

MIEF1 Microprotein Regulates Mitochondrial Translation.

Biochemistry 2018 09 14;57(38):5564-5575. Epub 2018 Sep 14.

Clayton Foundation Laboratories for Peptide Biology , The Salk Institute for Biological Studies , 10010 North Torrey Pines Road , La Jolla , California 92037 , United States.

Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.
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http://dx.doi.org/10.1021/acs.biochem.8b00726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443411PMC
September 2018

Antibiotic-induced microbiome depletion alters metabolic homeostasis by affecting gut signaling and colonic metabolism.

Nat Commun 2018 07 20;9(1):2872. Epub 2018 Jul 20.

Regulatory Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA, 92037, USA.

Antibiotic-induced microbiome depletion (AIMD) has been used frequently to study the role of the gut microbiome in pathological conditions. However, unlike germ-free mice, the effects of AIMD on host metabolism remain incompletely understood. Here we show the effects of AIMD to elucidate its effects on gut homeostasis, luminal signaling, and metabolism. We demonstrate that AIMD, which decreases luminal Firmicutes and Bacteroidetes species, decreases baseline serum glucose levels, reduces glucose surge in a tolerance test, and improves insulin sensitivity without altering adiposity. These changes occur in the setting of decreased luminal short-chain fatty acids (SCFAs), especially butyrate, and the secondary bile acid pool, which affects whole-body bile acid metabolism. In mice, AIMD alters cecal gene expression and gut glucagon-like peptide 1 signaling. Extensive tissue remodeling and decreased availability of SCFAs shift colonocyte metabolism toward glucose utilization. We suggest that AIMD alters glucose homeostasis by potentially shifting colonocyte energy utilization from SCFAs to glucose.
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http://dx.doi.org/10.1038/s41467-018-05336-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054678PMC
July 2018

Author Correction: Blocking Zika virus vertical transmission.

Sci Rep 2018 Jun 5;8(1):8794. Epub 2018 Jun 5.

University of California San Diego, School of Medicine, Department of Pediatrics/Rady Children's Hospital San Diego, Department of Cellular & Molecular Medicine, Stem Cell Program, La Jolla, CA, 92037-0695, USA.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-26959-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986756PMC
June 2018

The influence of transcript assembly on the proteogenomics discovery of microproteins.

PLoS One 2018 27;13(3):e0194518. Epub 2018 Mar 27.

Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, CA, United States of America.

Proteogenomics methods have identified many non-annotated protein-coding genes in the human genome. Many of the newly discovered protein-coding genes encode peptides and small proteins, referred to collectively as microproteins. Microproteins are produced through ribosome translation of small open reading frames (smORFs). The discovery of many smORFs reveals a blind spot in traditional gene-finding algorithms for these genes. Biological studies have found roles for microproteins in cell biology and physiology, and the potential that there exists additional bioactive microproteins drives the interest in detection and discovery of these molecules. A key step in any proteogenomics workflow is the assembly of RNA-Seq data into likely mRNA transcripts that are then used to create a searchable protein database. Here we demonstrate that specific features of the assembled transcriptome impact microprotein detection by shotgun proteomics. By tailoring transcript assembly for downstream mass spectrometry searching, we show that we can detect more than double the number of high-quality microprotein candidates and introduce a novel open-source mRNA assembler for proteogenomics (MAPS) that incorporates all of these features. By integrating our specialized assembler, MAPS, and a popular generalized assembler into our proteogenomics pipeline, we detect 45 novel human microproteins from a high quality proteogenomics dataset of a human cell line. We then characterize the features of the novel microproteins, identifying two classes of microproteins. Our work highlights the importance of specialized transcriptome assembly upstream of proteomics validation when searching for short and potentially rare and poorly conserved proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194518PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870951PMC
July 2018
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