The creation of a coherent genomic map of recent selection is one of the greatest challenges towards a better understanding of human evolution and the identification of functional genetic variants. Several methods have been proposed to detect linkage disequilibrium (LD), which is indicative of natural selection, from genome-wide profiles of common genetic variations but are designed for large regions.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1(+/-) mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1(+/+) and Becn1(+/-) immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.

The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTA(HDEL)) showed a similar cytotoxic effect on yeast as a corresponding RTA(KDEL) variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.

In yeast, apoptotic cell death can be triggered by various factors such as H2O2, cell aging, or acetic acid. Yeast caspase (Yca1p) and cellular reactive oxygen species (ROS) are key regulators of this process. Here, we show that moderate doses of three virally encoded killer toxins (K1, K28, and zygocin) induce an apoptotic yeast cell response, although all three toxins differ significantly in their primary killing mechanisms. In contrast, high toxin concentrations prevent the occurrence of an apoptotic cell response and rather cause necrotic, toxin-specific cell killing. Studies with Deltayca1 and Deltagsh1 deletion mutants indicate that ROS accumulation as well as the presence of yeast caspase 1 is needed for apoptosis in toxin-treated yeast cells. We conclude that in the natural environment of toxin-secreting killer yeasts, where toxin concentration is usually low, induction of apoptosis might play an important role in efficient toxin-mediated cell killing.

Human cancers can be classified based on gene signatures quantifying the degree of cell proliferation and tissue remodelling (PR). However, the specific factors that drive the increased tissue remodelling in tumours are not fully understood. Here we address this question using colorectal cancer as a case study.

Formate overflow coupled to mitochondrial oxidative metabolism\ has been observed in cancer cell lines, but whether that takes place in the tumor microenvironment is not known. Here we report the observation of serine catabolism to formate in normal murine tissues, with a relative rate correlating with serine levels and the tissue oxidative state. Yet, serine catabolism to formate is increased in the transformed tissue of in vivo models of intestinal adenomas and mammary carcinomas. The increased serine catabolism to formate is associated with increased serum formate levels. Finally, we show that inhibition of formate production by genetic interference reduces cancer cell invasion and this phenotype can be rescued by exogenous formate. We conclude that increased formate overflow is a hallmark of oxidative cancers and that high formate levels promote invasion via a yet unknown mechanism.

Cancer cells exhibit high rates of glycolysis and glutaminolysis. Glycolysis can provide energy and glutaminolysis can provide carbon for anaplerosis and reductive carboxylation to citrate. However, all these metabolic requirements could be in principle satisfied from glucose. Here we investigate why cancer cells do not satisfy their metabolic demands using aerobic biosynthesis from glucose. Based on the typical composition of a mammalian cell we quantify the energy demand and the OxPhos burden of cell biosynthesis from glucose. Our calculation demonstrates that aerobic growth from glucose is feasible up to a minimum doubling time that is proportional to the OxPhos burden and inversely proportional to the mitochondria OxPhos capacity. To grow faster cancer cells must activate aerobic glycolysis for energy generation and uncouple NADH generation from biosynthesis. To uncouple biosynthesis from NADH generation cancer cells can synthesize lipids from carbon sources that do not produce NADH in their catabolism, including acetate and the amino acids glutamate, glutamine, phenylalanine and tyrosine. Finally, we show that cancer cell lines have an OxPhos capacity that is insufficient to support aerobic biosynthesis from glucose. We conclude that selection for high rate of biosynthesis implies a selection for aerobic glycolysis and uncoupling biosynthesis from NADH generation.

Obesity is a risk factor for cardiovascular diseases, diabetes and cancer. In theory, the obesity problem could be solved by the adherence to a calorie-restricted diet, but that is not generally achieved in practice. An alternative is a pharmacological approach, using compounds that trigger the same metabolic changes associated with calorie restriction. Here, I expand in the pharmacological direction by identifying compounds that induce liver gene signature profiles that mimic those induced by calorie restriction. Using gene expression profiles from mice and rat, I identify corticosteroids, PPAR agonists and some antibacterial/antifungal as candidate compounds mimicking the response to calorie restriction in the liver gene signatures.

KDEL receptors (KDELRs) represent transmembrane proteins of the secretory pathway which regulate the retention of soluble ER-residents as well as retrograde and anterograde vesicle trafficking. In addition, KDELRs are involved in the regulation of cellular stress response and ECM degradation. For a deeper insight into KDELR1 specific functions, we characterised a KDELR1-KO cell line (HAP1) through whole transcriptome analysis by comparing KDELR1-KO cells with its respective HAP1 wild-type. Our data indicate more than 300 significantly and differentially expressed genes whose gene products are mainly involved in developmental processes such as cell adhesion and ECM composition, pointing out to severe cellular disorders due to a loss of KDELR1. Impaired adhesion capacity of KDELR1-KO cells was further demonstrated through in vitro adhesion assays, while collagen- and/or laminin-coating nearly doubled the adhesion property of KDELR1-KO cells compared to wild-type, confirming a transcriptional adaptation to improve or restore the cellular adhesion capability. Perturbations within the secretory pathway were verified by an increased secretion of ER-resident PDI and decreased cell viability under ER stress conditions, suggesting KDELR1-KO cells to be severely impaired in maintaining cellular homeostasis.

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

The vast majority of Parkinson's disease cases are idiopathic. Unclear etiology and multifactorial nature complicate the comprehension of disease pathogenesis. Identification of early transcriptomic and metabolic alterations consistent across different idiopathic Parkinson's disease (IPD) patients might reveal the potential basis of increased dopaminergic neuron vulnerability and primary disease mechanisms. In this study, we combine systems biology and data integration approaches to identify differences in transcriptomic and metabolic signatures between IPD patient and healthy individual-derived midbrain neural precursor cells. Characterization of gene expression and metabolic modeling reveal pyruvate, several amino acid and lipid metabolism as the most dysregulated metabolic pathways in IPD neural precursors. Furthermore, we show that IPD neural precursors endure mitochondrial metabolism impairment and a reduced total NAD pool. Accordingly, we show that treatment with NAD precursors increases ATP yield hence demonstrating a potential to rescue early IPD-associated metabolic changes.

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.

Carbon catabolite repression (CCR) is critical for optimal bacterial growth, and in bacterial (and yeast) cells it leads to their selective consumption of a single substrate from a complex environment. However, the root cause(s) for the development of this regulatory mechanism is unknown. Previously, a flux balance model (FBAwMC) of Escherichia coli metabolism that takes into account the crowded intracellular milieu of the bacterial cell correctly predicted selective glucose uptake in a medium containing five different carbon sources, suggesting that CCR may be an adaptive mechanism that ensures optimal bacterial metabolic network activity for growth.

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.

In mammals, new neurons are generated throughout the entire lifespan in two restricted areas of the brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ)-olfactory bulb (OB) system. In both regions newborn neurons display unique properties that clearly distinguish them from mature neurons. Enhanced excitability and increased synaptic plasticity enables them to add specific properties to information processing by modulating the existing local circuitry of already established mature neurons. Hippocampal neurogenesis has been suggested to play a role in spatial-navigation learning, spatial memory, and spatial pattern separation. Cumulative evidences implicate that adult-born OB neurons contribute to learning processes and odor memory. We recently demonstrated that the cell fate determinant TRIM32 is upregulated in differentiating neuroblasts of the SVZ-OB system in the adult mouse brain. The absence of TRIM32 leads to increased progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated OB neurons. Here, we present novel data from behavioral studies showing that such an enhancement of OB neurogenesis not necessarily leads to increased olfactory performance but in contrast even results in impaired olfactory capabilities. In addition, we show at the cellular level that TRIM32 protein levels increase during differentiation of neural stem cells (NSCs). At the molecular level, several metabolic intermediates that are connected to glycolysis, glycine, or cysteine metabolism are deregulated in TRIM32 knockout mice brain tissue. These metabolomics pathways are directly or indirectly linked to anxiety or depression like behavior. In summary, our study provides comprehensive data on how the impairment of neurogenesis caused by the loss of the cell fate determinant TRIM32 causes a decrease of olfactory performance as well as a deregulation of metabolomic pathways that are linked to mood disorders.

Aging is a complex trait of broad scientific interest, especially because of its intrinsic link with common human diseases. Pioneering work on aging-related mechanisms has been made in Saccharomyces cerevisiae, mainly through the use of deletion collections isogenic to the S288c reference strain. In this study, using a recently published high-throughput approach, we quantified chronological life span (CLS) within a collection of 58 natural strains across seven different conditions. We observed a broad aging variability suggesting the implication of diverse genetic and environmental factors in chronological aging control. Two major Quantitative Trait Loci (QTLs) were identified within a biparental population obtained by crossing two natural isolates with contrasting aging behavior. Detection of these QTLs was dependent upon the nature and concentration of the carbon sources available for growth. In the first QTL, the RIM15 gene was identified as major regulator of aging under low glucose condition, lending further support to the importance of nutrient-sensing pathways in longevity control under calorie restriction. In the second QTL, we could show that the SER1 gene, encoding a conserved aminotransferase of the serine synthesis pathway not previously linked to aging, is causally associated with CLS regulation, especially under high glucose condition. These findings hint toward a new mechanism of life span control involving a trade-off between serine synthesis and aging, most likely through modulation of acetate and trehalose metabolism. More generally it shows that genetic linkage studies across natural strains represent a promising strategy to further unravel the molecular basis of aging.

Like Ricin, Shiga, and Cholera toxins, yeast K28 is an A/B toxin that depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by these toxins remains incomplete. Since K28 is a model for clinically relevant toxins, we screened over 5000 yeast mutants, identifying 365 that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization, and cell wall biogenesis pathways. Furthermore, GPI anchors and transcriptional regulation are important for K28-cell binding. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. This comprehensive genetic analysis identified conserved processes important for A/B toxin trafficking and killing.

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.