Alan Saghatelian

Identification of protein pheromones that promote aggressive behaviour

Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male–male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Gαo subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male–male aggression through the accessory olfactory neural pathway.

Peptidomic discovery of short open reading frame–encoded peptides in human cells

The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that non-canonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8/1866) of long intergenic non-coding RNAs (lincRNAs). Together, these results provide the strongest evidence to date that the human proteome is more complex than previously appreciated.

Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects.

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.

A chiroselective peptide replicator.

The origin of homochirality in living systems is often attributed to the generation of enantiomeric differences in a pool of chiral prebiotic molecules, but none of the possible physiochemical processes considered can produce the significant imbalance required if homochiral biopolymers are to result from simple coupling of suitable precursor molecules. This implies a central role either for additional processes that can selectively amplify an initially minute enantiomeric difference in the starting material, or for a nonenzymatic process by which biopolymers undergo chiroselective molecular replication. Given that molecular self-replication and the capacity for selection are necessary conditions for the emergence of life, chiroselective replication of biopolymers seems a particularly attractive process for explaining homochirality in nature. Here we report that a 32-residue peptide replicator, designed according to our earlier principles, is capable of efficiently amplifying homochiral products from a racemic mixture of peptide fragments through a chiroselective autocatalytic cycle. The chiroselective amplification process discriminates between structures possessing even single stereochemical mutations within otherwise homochiral sequences. Moreover, the system exhibits a dynamic stereochemical ‘editing’ function; in contrast to the previously observed error correction, it makes use of heterochiral sequences that arise through uncatalysed background reactions to catalyse the production of the homochiral product. These results support the idea that self-replicating polypeptides could have played a key role in the origin of homochirality on Earth.

Toddler: An Embryonic Signal That Promotes Cell Movement via Apelin Receptors

Introduction Embryogenesis is thought to be directed by a small number of signaling pathways with most if not all embryonic signals having been identified. However, the molecular control of some embryonic processes is still poorly understood. For example, it is unclear how cell migration is regulated during gastrulation, when mesodermal and endodermal germ layers form. The goal of our study was to identify and characterize previously unrecognized signals that regulate embryogenesis. Toddler promotes gastrulation movements via Apelin receptor signaling. Toddler is an essential, short, conserved embryonic signal that promotes cell migration during zebrafish gastrulation. The internalization movement highlighted by the colored cell tracks requires Toddler signaling. Toddler signals via the G-protein–coupled APJ/Apelin receptor and may be one of several uncharacterized embryonic signals. Methods To identify uncharacterized signaling molecules, we mined zebrafish genomic data sets for previously non-annotated translated open reading frames (ORFs). One such ORF encoded a putative signaling protein that we call Toddler (also known as Apela/Elabela/Ende). We analyzed expression, production, and secretion of Toddler using RNA in situ hybridization, mass spectrometry, and Toddler-GFP fusion proteins, respectively. We used transcription activator-like effector (TALE) nucleases to generate frame-shift mutations in the toddler gene. To complement loss-of-function analyses with gain-of-function studies, Toddler was misexpressed through mRNA or peptide injection. We characterized phenotypes using marker gene expression analysis and in vivo imaging, using confocal and lightsheet microscopy. Toddler mutants were rescued thorugh global or localized toddler production. The relationship between Toddler and APJ/Apelin receptors was studied through genetic interaction and receptor internalization experiments. Results We identified several hundred non-annotated candidate proteins, including more than 20 putative signaling proteins. We focused on the functional importance of the short, conserved, and secreted peptide Toddler. Loss or overproduction of Toddler reduced cell movements during zebrafish gastrulation; mesodermal and endodermal cells were slow to internalize and migrate. Both the local and ubiquitous expression of Toddler were able to rescue gastrulation movements in toddler mutants, suggesting that Toddler acts as a motogen, a signal that promotes cell migration. Toddler activates G-protein–coupled APJ/Apelin receptor signaling, as evidenced by Toddler-induced internalization of APJ/Apelin receptors and rescue of toddler mutants through expression of the known receptor agonist Apelin. Discussion These findings indicate that Toddler promotes cell movement during zebrafish gastrulation by activation of APJ/Apelin receptor signaling. Toddler does not seem to act as a chemo-attractant or -repellent, but rather as a global signal that promotes the movement of mesendodermal cells. Both loss and overproduction of Toddler reduce cell movement, revealing that Toddler levels need to be tightly regulated during gastrulation. The discovery of Toddler helps explain previous genetic studies that found a broader requirement for APJ/Apelin receptors than for Apelin. We propose that in these cases, Toddler—not Apelin—activates APJ/Apelin receptor signaling. Our genomics analysis identifying a large number of candidate proteins that function during embryogenesis suggests the existence of other previously uncharacterized embryonic signals. Applying similar genomic approaches to adult tissues might identify additional signals that regulate physiological and behavioral processes. It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein–coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals. A conserved signal is identified that activates G protein–coupled receptors to promote zebrafish gastrulation. Toddler Welcome It has been assumed that most, if not all, major signals that control vertebrate embryogenesis have been identified. Using genomics, Pauli et al. (10.1126/science.1248636, published online 9 January) have now identified several new candidate signals expressed during early zebrafish development. One of these signals, Toddler, is a short, conserved, and secreted peptide that promotes the movement of cells during zebrafish gastrulation. Toddler signals through G protein–coupled receptors to drive internalization of the Apelin receptor, and activation of Apelin signaling can rescue toddler mutants.

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.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Exploring Disease through Metabolomics

Metabolomics approaches provide an analysis of changing metabolite levels in biological samples. In the past decade, technical advances have spurred the application of metabolomics in a variety of diverse research areas spanning basic, biomedical, and clinical sciences. In particular, improvements in instrumentation, data analysis software, and the development of metabolite databases have accelerated the measurement and identification of metabolites. Metabolomics approaches have been applied to a number of important problems, which include the discovery of biomarkers as well as mechanistic studies aimed at discovering metabolites or metabolic pathways that regulate cellular and physiological processes. By providing access to a portion of biomolecular space not covered by other profiling approaches (e.g., proteomics and genomics), metabolomics offers unique insights into small molecule regulation and signaling in biology. In the following review, we look at the integration of metabolomics approaches in different areas of basic and biomedical research, and try to point out the areas in which these approaches have enriched our understanding of cellular and physiological biology, especially within the context of pathways linked to disease.

An alternative pluripotent state confers interspecies chimaeric competency

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid use

Food intake increases the activity of hepatic de novo lipogenesis, which mediates the conversion of glucose to fats for storage or use. In mice, this program follows a circadian rhythm that peaks with nocturnal feeding and is repressed by Rev-erbα/β and an HDAC3-containing complex during the day. The transcriptional activators controlling rhythmic lipid synthesis in the dark cycle remain poorly defined. Disturbances in hepatic lipogenesis are also associated with systemic metabolic phenotypes, suggesting that lipogenesis in the liver communicates with peripheral tissues to control energy substrate homeostasis. Here we identify a PPARδ-dependent de novo lipogenic pathway in the liver that modulates fat use by muscle via a circulating lipid. The nuclear receptor PPARδ controls diurnal expression of lipogenic genes in the dark/feeding cycle. Liver-specific PPARδ activation increases, whereas hepatocyte-Ppard deletion reduces, muscle fatty acid uptake. Unbiased metabolite profiling identifies phosphatidylcholine 18:0/18:1 (PC(18:0/18:1) as a serum lipid regulated by diurnal hepatic PPARδ activity. PC(18:0/18:1) reduces postprandial lipid levels and increases fatty acid use through muscle PPARα. High-fat feeding diminishes rhythmic production of PC(18:0/18:1), whereas PC(18:0/18:1) administration in db/db mice (also known as Lepr−/−) improves metabolic homeostasis. These findings reveal an integrated regulatory circuit coupling lipid synthesis in the liver to energy use in muscle by coordinating the activity of two closely related nuclear receptors. These data implicate alterations in diurnal hepatic PPARδ–PC(18:0/18:1) signalling in metabolic disorders, including obesity.