Presynaptic sensor and silencer of peptidergic transmission reveal neuropeptides as primary transmitters in pontine fear circuit.

Neurons produce and release neuropeptides to communicate with one another. Despite their importance in brain function, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects presynaptic neuropeptide release and a genetically encoded silencer that specifically degrades neuropeptides inside LDCVs. Using these tools, we show that neuropeptides, not glutamate, encode the unconditioned stimulus in the parabrachial-to-amygdalar threat pathway during Pavlovian threat learning. We also show that neuropeptides play important roles in encoding positive valence and suppressing conditioned threat response in the amygdala-to-parabrachial endogenous opioidergic circuit. These results show that our sensor and silencer for presynaptic peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake, behaving animals.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases 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) have difficulty in colonizing 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 bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of 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.

Glucagon and Thyroid Hormone: A Championship Team.

A bioactive peptide that combines glucagon with the thyroid hormone T3 lowers lipid levels, improves glucose tolerance, and promotes energy expenditure to treat symptoms and underlying causes of metabolic disease. The two active components both maximize their combined benefits and mitigate the negative consequences of treatment with each alone.

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.

Primate-specific ORF0 contributes to retrotransposon-mediated diversity.

LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity.

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.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids.

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4-7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80-90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

Autoantibody mimicry of hormone action at the thyrotropin receptor.

Thyroid hormones are vital in metabolism, growth and development1. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)2. In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity3. How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains.

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

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 CHK1 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.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. 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 substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. 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.

A class of anti-inflammatory lipids decrease with aging in the central nervous system.

Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

Time-Restricted Feeding Reduces Atherosclerosis in LDLR KO Mice but Not in ApoE Knockout Mice.

BACKGROUND: Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS: We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS: TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating ß-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS: In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.

Mitochondrial DNA variation in Alzheimer's disease reveals a unique microprotein called SHMOOSE.

Mitochondrial DNA variants have previously associated with disease, but the underlying mechanisms have been largely elusive. Here, we report that mitochondrial SNP rs2853499 associated with Alzheimer's disease (AD), neuroimaging, and transcriptomics. We mapped rs2853499 to a novel mitochondrial small open reading frame called SHMOOSE with microprotein encoding potential. Indeed, we detected two unique SHMOOSE-derived peptide fragments in mitochondria by using mass spectrometry-the first unique mass spectrometry-based detection of a mitochondrial-encoded microprotein to date. Furthermore, cerebrospinal fluid (CSF) SHMOOSE levels in humans correlated with age, CSF tau, and brain white matter volume. We followed up on these genetic and biochemical findings by carrying out a series of functional experiments. SHMOOSE acted on the brain following intracerebroventricular administration, differentiated mitochondrial gene expression in multiple models, localized to mitochondria, bound the inner mitochondrial membrane protein mitofilin, and boosted mitochondrial oxygen consumption. Altogether, SHMOOSE has vast implications for the fields of neurobiology, Alzheimer's disease, and microproteins.

Pharmacological activation of REV-ERBs is lethal in cancer and oncogene-induced senescence.

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer and chronic circadian rhythm disruption predisposes individuals to tumour development. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERBα (also known as NR1D1) and REV-ERBß (also known as NR1D2), are essential components of the circadian clock. Here we show that two agonists of REV-ERBs-SR9009 and SR9011-are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.

A short ORF-encoded transcriptional regulator.

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.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

µProteInS-a proteogenomics pipeline for finding novel bacterial microproteins encoded by small ORFs.

SUMMARY: Genome annotation pipelines traditionally exclude open reading frames (ORFs) shorter than 100 codons to avoid false identifications. However, studies have been showing that these may encode functional microproteins with meaningful biological roles. We developed µProteInS, a proteogenomics pipeline that combines genomics, transcriptomics and proteomics to identify novel microproteins in bacteria. Our pipeline employs a model to filter out low confidence spectra, to avoid the need for manually inspecting Mass Spectrometry data. It also overcomes the shortcomings of traditional approaches that usually exclude overlapping genes, leaderless transcripts and non-conserved sequences, characteristics that are common among small ORFs (smORFs) and hamper their identification. AVAILABILITY AND IMPLEMENTATION: µProteInS is implemented in Python 3.8 within an Ubuntu 20.04 environment. It is an open-source software distributed under the GNU General Public License v3, available as a command-line tool. It can be downloaded at https://github.com/Eduardo-vsouza/uproteins and either installed from source or executed as a Docker image. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide.

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide 'small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.

Classical non-homologous end joining (cNHEJ) and homologous recombination compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

Defining the metabolome: size, flux, and regulation.

Renewed interest in metabolic research over the last two decades has inspired an explosion of technological developments for studying metabolism. At the forefront of methodological innovation is an approach referred to as "untargeted" or "discovery" metabolomics. The experimental objective of this technique is to comprehensively measure the entire metabolome, which constitutes a largely undefined set of molecules. Given its potential comprehensive coverage, untargeted metabolomics is often the first choice of experiments for investigators pursuing a metabolic research question. It is important to recognize, however, that untargeted metabolomics may not always be the optimal experimental approach. Conventionally, untargeted metabolomics only provides information about relative differences in metabolite pool sizes. Therefore, depending on the specific scientific question at hand, a complementary approach involving stable isotopes (such as metabolic flux analysis) may be better suited to provide biological insights. Unlike untargeted metabolomics, stable-isotope methods can provide information about differences in reaction rates.