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Alan Saghatelian

Publications and source records attributed to Alan Saghatelian.

16 recordsLinked to original sources
PubMed2025

Abnormal mitochondrial structure and function in brown adipose tissue of SLC35A4-MP knockout mice.

Uncovering the role of upstream open reading frames (uORFs) challenges conventional views of one protein per messenger RNA and reveals the capacity of some uORFs to encode microproteins that contribute to cellular biology and physiology. This study explores the functional role of a recently identified mitochondrial microprotein, SLC35A4-MP, in the brown adipose tissue of mice. Our findings reveal dynamic regulation of SLC35A4-MP expression during primary brown adipocyte differentiation in vitro and during cold exposure or high-fat diet (HFD)-induced obesity in mice. Using a knockout mouse model, we show that loss of SLC35A4-MP disrupts mitochondrial lipid composition, decreasing cardiolipins and phosphatidylethanolamine in brown adipose tissue from HFD-fed mice. SLC35A4-MP deficiency also impairs mitochondrial activity, alters mitochondrial number and morphology, and promotes inflammation. Knockout mice accumulate acylcarnitines during cold exposure, indicating defective fatty acid oxidation. These findings reveal SLC35A4-MP as a previously unrecognized microprotein in regulating mitochondrial function and tissue lipid metabolism, adding to the growing list of functional endogenous microproteins.

Animals↗
PubMed2006

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels.

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.

Amidohydrolases↗
PubMed2006

Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids.

N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. Like other lipid transmitters, NAEs are thought to be biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, we describe the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene [NAPE-PLD(-/-) mice]. Brain tissue from NAPE-PLD(-/-) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(-/-) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains (>or=C20). Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole.

Animals↗
PubMed2006

An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling.

Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.

Carbamates↗
PubMed2005

Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling.

Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function. However, determining the protein targets of bioactive compounds remains a formidable challenge. We address this problem here through the creation of a natural product-inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

Antineoplastic Agents↗
PubMed2005

Discovery metabolite profiling--forging functional connections between the proteome and metabolome.

Of primary interest for every enzyme is the identification of its physiological substrates. However, the vast structural diversity of endogenous metabolites, coupled with the overlapping activities of numerous enzymes, makes it difficult to deduce the identity of natural substrates for a given enzyme based on in vitro experiments. To address this challenge, we recently introduced an LC-MS based analytical method termed discovery metabolite profiling (DMP) to evaluate the global metabolic effects of enzyme inactivation in vivo. We have applied DMP to study mice lacking the enzyme fatty acid amide hydrolase (FAAH), which degrades the endocannabinoid family of signaling lipids. DMP identified several previously uncharacterized FAAH substrates, including a structurally novel class of brain lipids that represent conjugates of very long chain fatty acids with the amino acid derivative taurine [N-acyl taurines (NATs)]. These findings show that DMP can establish direct connections between the proteome and metabolome and thus offers a powerful strategy to assign physiological functions to enzymes in the post-genomic era.

Amidohydrolases↗
PubMed2005

Global strategies to integrate the proteome and metabolome.

A fundamental goal of proteomics is to assign physiological functions to all proteins encoded by eukaryotic and prokaryotic genomes. Of the many activities performed by proteins, the chemical transformations catalyzed by enzymes form the basis for most, if not all, metabolic and signaling pathways. Elucidation of these pathways and their integration into larger cellular networks require new strategies to rapidly and systematically identify physiological substrates of enzymes. Here, we review emerging technologies that aim to assign endogenous biochemical functions to enzymes by profiling the metabolome.

Animals↗
PubMed2005

Assignment of protein function in the postgenomic era.

Genome sequencing projects have provided researchers with an unprecedented boon of molecular information that promises to revolutionize our understanding of life and lead to new treatments of its disorders. However, genome sequences alone offer only limited insights into the biochemical pathways that determine cell and tissue function. These complex metabolic and signaling networks are largely mediated by proteins. The vast number of uncharacterized proteins found in prokaryotic and eukaryotic systems suggests that our knowledge of cellular biochemistry is far from complete. Here, we highlight a new breed of 'postgenomic' methods that aim to assign functions to proteins through the integrated application of chemical and biological techniques.

Amino Acid Sequence↗
PubMed2004

Assignment of endogenous substrates to enzymes by global metabolite profiling.

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.

Amidohydrolases↗
PubMed2004

Functional disassociation of the central and peripheral fatty acid amide signaling systems.

Fatty acid amides (FAAs) constitute a large class of endogenous signaling lipids that modulate several physiological processes, including pain, feeding, blood pressure, sleep, and inflammation. Although FAAs have been proposed to evoke their behavioral effects through both central and peripheral mechanisms, these distinct signaling pathways have remained experimentally challenging to separate. Here, we report a transgenic mouse model in which the central and peripheral FAA systems have been functionally uncoupled. Mice were generated that express the principle FAA-degrading enzyme FAA hydrolase (FAAH) specifically in the nervous system (FAAH-NS mice) by crossing FAAH(-/-) mice with transgenic mice that express FAAH under the neural specific enolase promoter. FAAH-NS mice were found to possess wild-type levels of FAAs in the brain and spinal cord, but significantly elevated concentrations of these lipid transmitters in peripheral tissues. This anatomically restricted biochemical phenotype correlated with a reversion of the reduced pain sensitivity of FAAH(-/-) mice, consistent with the FAA anandamide producing this effect by acting on cannabinoid receptors in the nervous system. Interestingly, however, FAAH-NS mice still exhibited an antiinflammatory phenotype similar in magnitude to FAAH(-/-) mice, indicating that this activity, which was not blocked by cannabinoid receptor antagonists, was mediated by peripherally elevated FAAs. These data suggest that the central and peripheral FAA signaling systems regulate discrete behavioral processes and may be targeted for distinct therapeutic gain.

Amides↗
PubMed2004

Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity.

Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.

Amidohydrolases↗
PubMed2004

Activity-based probes for the proteomic profiling of metalloproteases.

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

Dipeptides↗
PubMed2003

DNA detection and signal amplification via an engineered allosteric enzyme.

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.

Bacillus cereus↗
PubMed2003

DNA-based photonic logic gates: AND, NAND, and INHIBIT.

Conventional microprocessors use elementary logic gates to perform complex computational tasks. Mimicking such computational processes using purely molecular systems has been limited in most cases by the lack of design generality or potential addressability of existing molecular logic gates. Herein we report that by employing the universal recognition properties of DNA simple photonic logic gates can be created that are capable of AND, NAND, and INHIBIT logic operations.

Computational Biology↗