Estrogen receptor-related receptor (Esrra) induces ribosomal protein Rplp1-mediated adaptive hepatic translation during prolonged starvation.

Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.

Discovering microproteins: making the most of ribosome profiling data.

Building a reference set of protein-coding open reading frames (ORFs) has revolutionized biological process discovery and understanding. Traditionally, gene models have been confirmed using cDNA sequencing and encoded translated regions inferred using sequence-based detection of start and stop combinations longer than 100 amino-acids to prevent false positives. This has led to small ORFs (smORFs) and their encoded proteins left un-annotated. Ribo-seq allows deciphering translated regions from untranslated irrespective of the length. In this review, we describe the power of Ribo-seq data in detection of smORFs while discussing the major challenge posed by data-quality, -depth and -sparseness in identifying the start and end of smORF translation. In particular, we outline smORF cataloguing efforts in humans and the large differences that have arisen due to variation in data, methods and assumptions. Although current versions of smORF reference sets can already be used as a powerful tool for hypothesis generation, we recommend that future editions should consider these data limitations and adopt unified processing for the community to establish a canonical catalogue of translated smORFs.

LINC00116-encoded microprotein mitoregulin regulates fatty acid metabolism at the mitochondrial outer membrane.

LINC00116 encodes a microprotein first identified as Mitoregulin (MTLN), where it was reported to localize to the inner membrane of mitochondria to regulate fatty acid oxidation and oxidative phosphorylation. These initial discoveries were followed by reports with differing findings about its molecular functions and submitochondrial localization. To clarify the apparent discrepancies, we constructed multiple orthogonal methods of determining the localization of MTLN, including split GFP-based reporters that enable efficient and reliable topology analyses for microproteins. These methods unequivocally demonstrate MTLN primarily localizes to the outer membrane of mitochondria, where it interacts with enzymes of fatty acid metabolism including CPT1B and CYB5B. Loss of MTLN causes the accumulation of very long-chain fatty acids (VLCFAs), especially docosahexaenoic acid (DHA). Intriguingly, loss of MTLN protects mice against western diet/fructose-induced insulin-resistance, suggests a protective effect of VLCFAs in this context. MTLN thus serves as an attractive target to control the catabolism of VLCFAs.

Mast cell activation in lungs during SARS-CoV-2 infection associated with lung pathology and severe COVID-19.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in patients who are severely ill, and the pathophysiology of disease is thought to be immune mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens and often promote inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and nonhuman primates. Using a mouse model of MC deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed through detection of MC-specific proteases, including chymase, the levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.

Spns1 is a lysophospholipid transporter mediating lysosomal phospholipid salvage.

The lysosome is central to the degradation of proteins, carbohydrates, and lipids and their salvage back to the cytosol for reutilization. Lysosomal transporters for amino acids, sugars, and cholesterol have been identified, and the metabolic fates of these molecules in the cytoplasm have been elucidated. Remarkably, it is not known whether lysosomal salvage exists for glycerophospholipids, the major constituents of cellular membranes. By using a transport assay screen against orphan lysosomal transporters, we identified the major facilitator superfamily protein Spns1 that is ubiquitously expressed in all tissues as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, with LPC and LPE being the lysosomal breakdown products of the most abundant eukaryotic phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively. Spns1 deficiency in cells, zebrafish embryos, and mouse liver resulted in lysosomal accumulation of LPC and LPE species with pathological consequences on lysosomal function. Flux analysis using stable isotope-labeled phospholipid apolipoprotein E nanodiscs targeted to lysosomes showed that LPC was transported out of lysosomes in an Spns1-dependent manner and re-esterified back into the cytoplasmic pools of phosphatidylcholine. Our findings identify a phospholipid salvage pathway from lysosomes to the cytosol that is dependent on Spns1 and critical for maintaining normal lysosomal function.

Viral proteases activate the CARD8 inflammasome in the human cardiovascular system.

Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.

Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis.

Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

CLIPreg: constructing translational regulatory networks from CLIP-, Ribo- and RNA-seq.

MOTIVATION: The creation and analysis of gene regulatory networks have been the focus of bioinformatics research and underpins much of what is known about gene regulation. However, as a result of a bias in the availability of data types that are collected, the vast majority of gene regulatory network resources and tools have focused on either transcriptional regulation or protein-protein interactions. This has left other areas of regulation, for instance, translational regulation, vastly underrepresented despite them having been shown to play a critical role in both health and disease. RESULTS: In order to address this, we have developed CLIPreg, a package that integrates RNA, Ribo and CLIP- sequencing data in order to construct translational regulatory networks coordinated by RNA-binding proteins and micro-RNAs. This is the first tool of its type to be created, allowing for detailed investigation into a previously unseen layer of regulation. AVAILABILITY AND IMPLEMENTATION: CLIPreg is available at https://github.com/SGDDNB/CLIPreg. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

Signatures of mast cell activation are associated with severe COVID-19.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.

Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named "Modulator of cytochrome C oxidase during Inflammation" (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation.

sORF-Encoded MicroPeptides: New players in inflammation, metabolism, and precision medicine.

Significant technological advances have enabled the discovery and identification of a new class of molecules, micropeptides or small ORF encoded peptides (SEPs) within non-coding RNAs (ncRNAs). As ncRNAs are well known to be transcriptionally silent, the discovery of SEPs implies that many ncRNAs are misannotated or play both coding and non-coding functions. SEPs have reportedly diverse regulatory roles in embryogenesis, myogenesis, inflammation, diseases, and cancer. SEPs appearing in different subcellular compartments show distinct functions. In this review, we summarized the functions of SEPs that have been characterized thus far. As SEPs are amenable to therapeutic development as biologics, understanding their underlying functions will provide novel targets for the treatment of inflammatory or metabolic disorders.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Alternative Progenitor Cells Compensate to Rebuild the Coronary Vasculature in Elabela- and Apj-Deficient Hearts.

Organogenesis during embryonic development occurs through the differentiation of progenitor cells. This process is extraordinarily accurate, but the mechanisms ensuring high fidelity are poorly understood. Coronary vessels of the mouse heart derive from at least two progenitor pools, the sinus venosus and endocardium. We find that the ELABELA (ELA)-APJ signaling axis is only required for sinus venosus-derived progenitors. Because they do not depend on ELA-APJ, endocardial progenitors are able to expand and compensate for faulty sinus venosus development in Apj mutants, leading to normal adult heart function. An upregulation of endocardial SOX17 accompanied compensation in Apj mutants, which was also seen in Ccbe1 knockouts, indicating that the endocardium is activated in multiple cases where sinus venosus angiogenesis is stunted. Our data demonstrate that by diversifying their responsivity to growth cues, distinct coronary progenitor pools are able to compensate for each other during coronary development, thereby providing robustness to organ development.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

ELABELA-APJ axis protects from pressure overload heart failure and angiotensin II-induced cardiac damage.

AIMS: Elabela/Toddler/Apela (ELA) has been identified as a novel endogenous peptide ligand for APJ/Apelin receptor/Aplnr. ELA plays a crucial role in early cardiac development of zebrafish as well as in maintenance of self-renewal of human embryonic stem cells. Apelin was the first identified APJ ligand, and exerts positive inotropic heart effects and regulates the renin-angiotensin system. The aim of this study was to investigate the biological effects of ELA in the cardiovascular system. METHODS AND RESULTS: Continuous infusion of ELA peptide significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and impaired contractility in mice. ELA treatment reduced mRNA expression levels of genes associated with heart failure and fibrosis. The cardioprotective effects of ELA were diminished in APJ knockout mice, indicating that APJ is the key receptor for ELA in the adult heart. Mechanistically, ELA downregulated angiotensin-converting enzyme (ACE) expression in the stressed hearts, whereas it showed little effects on angiotensin-converting enzyme 2 (ACE2) expression, which are distinct from the effects of Apelin. FoxM1 transcription factor, which induces ACE expression in the stressed hearts, was downregulated by ELA but not by Apelin. ELA antagonized angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. CONCLUSION: The ELA-APJ axis protects from pressure overload-induced heart failure possibly via suppression of ACE expression and pathogenic angiotensin II signalling. The different effects of ELA and Apelin on the expression of ACE and ACE2 implicate fine-tuned mechanisms for a ligand-induced APJ activation and downstream signalling.