Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike.

The global spread of the SARS-CoV-2 virus has resulted in emergence of lineages which impact the effectiveness of immunotherapies and vaccines that are based on the early Wuhan isolate. All currently approved vaccines employ the spike protein S, as it is the target for neutralizing antibodies. Here we describe two SARS-CoV-2 isolates with unusually large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis shows that the deletions result in complete reshaping of the NTD supersite, an antigenically important region of the NTD. For both spike variants the remodeling of the NTD negatively affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite. For one of the variants, we observed a P9L mediated shift of the signal peptide cleavage site resulting in the loss of a disulfide-bridge; a unique escape mechanism with high antigenic impact. Although the observed deletions and disulfide mutations are rare, similar modifications have become independently established in several other lineages, indicating a possibility to become more dominant in the future. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment.

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.

Corrigendum: Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

This corrects the article DOI: 10.1038/nature22056.

Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli.

Carbapenems, a family of ß-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism.

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.

The serine/threonine phosphatase PPM1B (PP2Cß) selectively modulates PPARγ activity.

Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cß] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.

Multi-omics & pathway analysis identify potential roles for tumor N-acetyl aspartate accumulation in murine models of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge (Oakes and Papa, 2015). Matched untargeted multi-level omic datasets may reveal biological changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics were performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node, and bone metastasis grown as murine orthografts. Collectively, we tested the feasibility of muti-omics analysis on models of CRPC in revealing pathways of interest for future validation investigation. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-acetylated alpha-linked acidic dipeptidases (FOLH1/NAALADL2). Based on pathway analysis integrating multiple omic levels, we hypothesize that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of acetyl Co-A for upregulated sphingolipid metabolism and a pool of glutamate and aspartate for nucleotide synthesis during tumor growth.

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response.

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27(kip1) gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27(kip1) transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27(kip1) transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70(-/-) ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 µM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.

Calibrating the Bacterial Growth Rate Speedometer: A Re-evaluation of the Relationship Between Basal ppGpp, Growth, and RNA Synthesis in Escherichia coli.

The molecule guanosine tetraphophosphate (ppGpp) is most commonly considered an alarmone produced during acute stress. However, ppGpp is also present at low concentrations during steady-state growth. Whether ppGpp controls the same cellular targets at both low and high concentrations remains an open question and is vital for understanding growth rate regulation. It is widely assumed that basal ppGpp concentrations vary inversely with growth rate, and that the main function of basal ppGpp is to regulate transcription of ribosomal RNA in response to environmental conditions. Unfortunately, studies to confirm this relationship and to define regulatory targets of basal ppGpp are limited by difficulties in quantifying basal ppGpp. In this Perspective we compare reported concentrations of basal ppGpp in E. coli and quantify ppGpp within several strains using a recently developed analytical method. We find that although the inverse correlation between ppGpp and growth rate is robust across strains and analytical methods, absolute ppGpp concentrations do not absolutely determine RNA synthesis rates. In addition, we investigated the consequences of two separate RNA polymerase mutations that each individually reduce (but do not abolish) sensitivity to ppGpp and find that the relationship between ppGpp, growth rate, and RNA content of single-site mutants remains unaffected. Both literature and our new data suggest that environmental conditions may be communicated to RNA polymerase via an additional regulator. We conclude that basal ppGpp is one of potentially several agents controlling ribosome abundance and DNA replication initiation, but that evidence for additional roles in controlling macromolecular synthesis requires further study.

Posttranslational Control of PlsB Is Sufficient To Coordinate Membrane Synthesis with Growth in Escherichia coli.

Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis.IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth.

Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex.

Efficient transcription is linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptional activity and is regulated by the Bur1/2 kinase complex. We found that the evolutionarily conserved Ccr4-Not complex is involved in establishing H3K4 tri-methylation in Saccharomyces cerevisiae. We observed synthetic lethal interactions of Ccr4-Not components with BUR1 and BUR2. Further analysis indicated that the genes encoding the Not-proteins are essential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 or H3K79me2/3 levels. Moreover, regulation of H3K4me3 levels by NOT4 is independent of defects in RNA polymerase II loading. We found NOT4 to be important for ubiquitylation of histone H2B via recruitment of the PAF complex, but not for recruitment or activation of the Bur1/2 complex. These results suggest a mechanism in which the Ccr4-Not complex functions parallel to or downstream of the Bur1/2 kinase to facilitate H3K4me3 via PAF complex recruitment.

The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma.

The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip60 protein is recruited to PPARgamma target genes in mature 3T3-L1 adipocytes but not in preadipocytes, indicating that Tip60 requires PPARgamma for its recruitment to PPARgamma target genes. Importantly, we show that in common with disruption of PPARgamma function, small interfering RNA-mediated reduction of Tip60 protein impairs differentiation of 3T3-L1 preadipocytes. Taken together, these findings qualify the acetyltransferase Tip60 as a novel adipogenic factor.

Analysis of Cell Metabolism Using LC-MS and Isotope Tracers.

Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using (13)C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for (13)C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism.

Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis.

Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress.

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.

Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells.

Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine--rather than glycine--consumption.

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner.

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD+-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis.

A protocol for quantifying lipid peroxidation in cellular systems by F2-isoprostane analysis.

Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF2α-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems.

Phosphorylation of Not4p functions parallel to BUR2 to regulate resistance to cellular stresses in Saccharomyces cerevisiae.

BACKGROUND: The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p. METHODOLOGY/PRINCIPAL FINDINGS: First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Delta strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Delta strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. CONCLUSIONS: Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in Saccharomyces cerevisiae.