Biology subjects

Saghatelian, A.

Publications and source records attributed to Saghatelian, A..

18 recordsLinked to original sources

bioRxiv2025

CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism

Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation. SignificanceObesity and its associated metabolic disorders pose significant public health challenges, yet the molecular mechanisms regulating adipocyte function remain incompletely understood. Small open reading frames (smORFs) and their encoded microproteins represent an emerging class of regulatory elements with potential roles in metabolism. Here, we leveraged CRISPR/Cas9 screening to functionally characterize smORFs in adipocytes, identifying novel regulators of cell proliferation and lipid metabolism. Our findings demonstrate that conservation is not a prerequisite for smORF function, as we validated a mouse-specific microprotein that modulates adipocyte differentiation. This work establishes a robust pipeline for unbiased smORF discovery and highlights the potential for species-specific microproteins to regulate adipose biology. Future studies in human adipocytes may uncover additional microproteins with therapeutic relevance for obesity and metabolic disease.

cell biology↗

bioRxiv2025

Trifluoroacetate reduces plasma lipid levels and the development of atherosclerosis in mice

Trifluoroacetate (TFA) has been assumed to be an innocuous counterion (to cationic amino acid side chains) present in countless synthetic bioactive peptides and a few FDA-approved therapeutics. We show here that TFA is in fact bioactive and causes dramatic biological effects in multiple strains of mice and cultured human and rat liver cells. In high-fat diet (HFD)-fed low-density lipoprotein receptor-null (LDLr-/-) mice, TFA reduces the levels of plasma cholesterol, triglycerides, and the development of atherosclerotic lesions following either oral or intraperitoneal administration. These physiological effects were observed with TFA alone, or with TFA present as a counterion of a variety of short, unrelated synthetic peptide sequences. Mechanistic investigations including RNA-seq, confocal microscopy, western blotting, metabolomics, proteomics, pharmacokinetics, and biochemical assays indicated that TFA induces peroxisome proliferation by activating peroxisome proliferator-activated receptor (PPAR)-alpha. We confirmed that TFA also caused peroxisome proliferation and downstream phenotypic effects in cultured human and rat liver cells, wild-type C57/Bl mice, and apolipoprotein E-null (apoE-/-) mice, leading to anti-atherosclerotic effects in the latter strain. Given that TFA is a counterion in many peptides employed in early research and development settings, these findings raise the possibility that TFA may be confounding or contributing to phenotypic changes observed in many studies involving peptides. Although our studies suggest that TFA or its analogues might have therapeutic applications, it should be noted that TFA is also a persistent environmental contaminant that is found at high levels in humans relative to other polyfluoroalkyl substances (PFAS), and is a major metabolite following treatment of patients with common inhaled anesthetics, suggesting that the biological effects reported here could have other implications for human health.

biochemistry↗

bioRxiv2024

Dual quorum-sensing control of purine biosynthesis drives pathogenic fitness of Enterococcus faecalis

Enterococcus faecalis is a resident of the human gut, though upon translocation to the blood or body tissues, it can be pathogenic. Here we discover and characterize two peptide-based quorum-sensing systems that transcriptionally modulate de novo purine biosynthesis in E. faecalis. Using a comparative genomic analysis, we find that most enterococcal species do not encode this system; E. moraviensis, E. haemoperoxidus and E. caccae, three species that are closely related to E. faecalis, encode one of the two systems, and only E. faecalis encodes both systems. We show that these systems are important for the intracellular survival of E. faecalis within macrophages and for the fitness of E. faecalis in a murine wound infection model. Taken together, we combine comparative genomics, microbiological, bacterial genetics, transcriptomics, targeted proteomics and animal model experiments to describe a paired quorum sensing mechanism that directly influences central metabolism and impacts the pathogenicity of E. faecalis.

microbiology↗

bioRxiv2023

Large-scale proteogenomics characterization of the Mycobacterium tuberculosis hidden microproteome

Tuberculosis remains a burden to this day, due to the rise of multi and extensively drug-resistant bacterial strains. The genome of Mycobacterium tuberculosis (Mtb) underwent an annotation process that excluded small Open Reading Frames (smORFs), which encode a class of peptides and small proteins collectively known as microproteins. As a result, there is an overlooked part of its proteome that is a rich source of potentially essential, druggable molecular targets. Here, we employed our recently developed proteogenomics pipeline to identify novel microproteins encoded by smORFs in the genome of Mtb usings hundreds of mass spectrometry experiments in a large-scale approach. We found protein evidence for hundreds of novel microproteins and identified smORFs potentially involved in bacterial growth and virulence. Moreover, many smORFs are co-expressed or share operons with a myriad of biologically relevant genes and may play a role in antibiotic response. Together, our data presents a resource of unknown genes that play a role in the success of Mtb as a widespread pathogen.

molecular biology↗

bioRxiv2023

Tissue-specific sex difference in the metabolism of fatty acid esters of hydroxy fatty acids

Fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous bioactive lipids known for their anti-inflammatory and anti-diabetic properties. Despite their therapeutic potential, little is known about the sex-specific variations in FAHFA metabolism. This study investigated the role of Androgen Dependent TFPI Regulating Protein (ADTRP), a FAHFA hydrolase. Additionally, tissue-specific differences in FAHFA levels, focusing on the perigonadal white adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), plasma, and liver, were evaluated using metabolomics and lipidomics. We found that female mice exhibited higher FAHFA levels in pgWAT, scWAT, and BAT compared to males. FAHFA levels were inversely related to Adtrp mRNA, which showed significantly lower expression in females compared with males in pgWAT and scWAT. However, no significant differences between the sexes were observed in plasma and liver FAHFA levels. Adtrp deletion had minimal impact on both sexes metabolome and lipidome of pgWAT. However, we discovered higher endogenous levels of triacylglycerol estolides containing FAHFAs, a FAHFA metabolic reservoir, in the pgWAT of female mice. These findings suggest that sex-dependent differences in FAHFA levels occur primarily in specific WAT depots and may modulate local insulin sensitivity in adipocytes. However, further investigations are warranted to fully comprehend the underlying mechanisms and implications of sex effects on FAHFA metabolism in humans.

biochemistry↗

bioRxiv2023

The Integration of Proteogenomics and Ribosome Profiling Circumvents Key Limitations to Increase the Coverage and Confidence of Novel Microproteins

There has been a dramatic increase in the identification of non-conical translation and a significant expansion of the protein-coding genome and proteome. Among the strategies used to identify novel small ORFs (smORFs), Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple sites in the genome are computationally removed since they cannot unambiguously be assigned to a specific genomic location, or to a specific transcript in the case of multiple isoforms. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of ambiguous and multi-mapping alignments, such that smORFs that reside in these regions cannot be identified by Ribo-Seq. Here, we show that the inclusion of proteogenomics to create a Ribosome Profiling and Proteogenomics Pipeline (RP3) bypasses this limitation to identify a group of microprotein-encoding smORFs that are missed by current Ribo-Seq pipelines. Moreover, we show that the microproteins identified by RP3 have different sequence compositions from the ones identified by Ribo-Seq-only pipelines, which can affect proteomics identification. In aggregate, the development of RP3 maximizes the detection and confidence of protein-encoding smORFs and microproteins.

bioinformatics↗

bioRxiv2023

Inflammation causes insulin resistance via interferon regulatory factor 3 (IRF3)-mediated reduction in FAHFA levels

Obesity-induced inflammation causes metabolic dysfunction, but the mechanisms remain elusive. Here we show that the innate immune transcription factor interferon regulatory factor (IRF3) adversely affects glucose homeostasis through induction of the endogenous FAHFA hydrolase androgen induced gene 1 (AIG1) in adipocytes. Adipocyte-specific knockout of IRF3 protects mice against high-fat diet-induced insulin resistance, whereas overexpression of IRF3 or AIG1 in adipocytes promotes insulin resistance on a high-fat diet. Furthermore, pharmacological inhibition of AIG1 reversed obesity-induced insulin resistance and restored glucose homeostasis in the setting of adipocyte IRF3 overexpression. We, therefore, identify the adipocyte IRF3/AIG1 axis as a crucial link between obesity-induced inflammation and insulin resistance and suggest an approach for limiting the metabolic dysfunction accompanying obesity.

physiology↗

bioRxiv2022

Proteomic discovery of chemical probes that perturb protein complexes in human cells

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

biochemistry↗

bioRxiv2022

Profiling Mouse Brown and White Adipocytes to Identify Metabolically Relevant Small ORFs and Functional Microproteins

The absence of thousands of recently annotated small open reading frame (smORF)-encoded peptides and small proteins (microproteins) from databases has precluded their analysis in metabolism and metabolic disease. Given the outsized importance of small proteins and peptides such as insulin, leptin, amylin, glucagon, and glucagon-like peptide-1 (GLP-1) in metabolism, microproteins are a potentially rich source of uncharacterized metabolic regulators. Here, we annotate smORFs in primary differentiated brown, white, and beige mouse adipose cells. Ribosome profiling (Ribo-Seq) detected a total of 3,877 unannotated smORFs. Analysis of RNA-Seq datasets revealed diet-regulated smORF expression in adipose tissues, and validated the adipose translation of the feeding-neuron marker gene Gm8773. Gm8773 encodes the mouse homolog of FAM237B, a neurosecretory protein that stimulates food intake and promotes weight gain in chickens. Testing of recombinant mFAM237B produced similar orexigenic activity in mice further supporting a role for FAM237B as a metabolic regulator and potentially part of the brain-adipose axis. Furthermore, we demonstrated that data independent acquisition mass spectrometry (DIA-MS) proteomics can provide a sensitive, flexible, and quantitative platform for identifying microproteins by mass spectrometry. Using this system led to the detection of 58 microproteins from cell culture and an additional 33 from mouse plasma. The proteomics data established the anti-inflammatory microprotein AW112010 as a circulating factor, and found that plasma levels of a microprotein translated from a FRS2 uORF is elevated in older obese mice. Together, the data highlight the value of this database in examining understudied smORFs and microproteins in metabolic research and identifying additional regulators of metabolism.

systems biology↗

bioRxiv2022

Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.

biochemistry↗

bioRxiv2021

Intestinal Transgene Delivery with Native E. coli Chassis Allows Persistent Physiological Changes

Live bacterial therapeutics (LBT) could reverse disease by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally-raised (CR) hosts have been unsuccessful, because engineered microbial organisms (i.e., chassis) cannot colonize the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli isolated from stool cultures of CR mice were modified to express functional bacterial (bile salt hydrolase) and eukaryotic (Interleukin-10) genes. Reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect host physiology and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock-in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts, and enables LBT with curative intent.

synthetic biology↗

bioRxiv2021

A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational vocabulary of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

genomics↗

bioRxiv2021

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

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 co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples 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 were 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 provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS. SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

cell biology↗

bioRxiv2020

ELOVL2 is required for robust visual function in zebrafish

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) play critical roles in membrane stability and cell signaling within the retina. Elovl2, an elongase involved in 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 elovl2 in retina development in embryonic zebrafish. Whole mount in situ hybridization shows elovl2 is expressed in the Muller glia in embryonic and adult zebrafish. Lipidomics analysis of elovl2 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 elovl2 revealed increased retinal thickness compared to controls at day 7 without gross disruptions of retinal architecture. Finally, elovl2 crispants showed differences in the visual motor reflex light off (VMR-OFF) at day 7 compared to controls. In sum, inactivation of elovl2 in zebrafish embryos caused changes in lipid composition and in visual behavior further confirming the important role of LC-PUFAs in healthy vision.

molecular biology↗

bioRxiv2019

The Lipid Elongation Enzyme ELOVL2 is a molecular regulator of aging in the retina

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 of complement system and lipid metabolism. These findings indicate that ELOVL2 activity regulates aging in mouse retina, provide a molecular link between polyunsaturated fatty acids elongation and visual functions, and suggest novel therapeutic strategies for treatment of age-related eye diseases.

molecular biology↗

bioRxiv2019

A novel role for Myosin-Va in mitochondrial fission

In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC=\"FIGDIR/small/655803v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (35K):\norg.highwire.dtl.DTLVardef@1130e72org.highwire.dtl.DTLVardef@196619org.highwire.dtl.DTLVardef@17e09edorg.highwire.dtl.DTLVardef@1bdc311_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

genomics↗

bioRxiv2017

TARGETED SEARCHES FOR NOVEL PEPTIDES IN BIG MASS SPECTROMETRY DATA SETS

We present Post-Acquisition Targeted Searches (PATS), an easy-to-use tool that allows the identification of novel peptide/protein sequences from existing big mass spectrometry data sets. PATS filters out the unrelated peptidome before the time-consuming database search to significantly speed up the identification. Using interactome data sets, PATS visualizes protein interaction network and helps to assign putative functions to the target protein based on the \"guilt by association\" concept.

bioinformatics↗