Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl–coenzyme A hydratase/3-hydroxyacyl–coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

The gut microbiota benefits humans via short-chain fatty acid (SCFA) production from carbohydrate fermentation, and deficiency in SCFA production is associated with type 2 diabetes mellitus (T2DM). We conducted a randomized clinical study of specifically designed isoenergetic diets, together with fecal shotgun metagenomics, to show that a select group of SCFA-producing strains was promoted by dietary fibers and that most other potential producers were either diminished or unchanged in patients with T2DM. When the fiber-promoted SCFA producers were present in greater diversity and abundance, participants had better improvement in hemoglobin A1c levels, partly via increased glucagon-like peptide-1 production. Promotion of these positive responders diminished producers of metabolically detrimental compounds such as indole and hydrogen sulfide. Targeted restoration of these SCFA producers may present a novel ecological approach for managing T2DM.

Cullins assemble the largest family of ubiquitin ligases by binding with ROC1 and various substrate receptors. CUL4 function is linked with many cellular processes, but its substrate-recruiting mechanism remains elusive. We identified a protein motif, the DWD box (DDB1-binding WD40 protein), and demonstrated the binding of 15 DWD proteins with DDB1-CUL4A. We provide evidence supporting the critical function of the DWD box and DDB1's role as the linker mediating DWD protein association with CUL4A. A database search predicts that about one-third of WD40 proteins, 90 in humans, contain the DWD box, suggesting a potentially large number of DWD-DDB1-CUL4-ROC1 E3 ligases. The ubiquitin-proteasome pathway regulates the concentration and conformation of many cellular proteins in response to changes in physiological conditions. This pathway consists of a cascade of three activities performed by E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase) enzymes (Hochstrasser 1996;King et al. 1996;Hershko and Ciechanover 1998). A critical step in this process is how individual protein substrates are recruited to specific E3 ligases. The RING family represents the major family of E3 ligases. Members either contain an intrinsic RING finger domain (as in MDM2 and BRCA1) or bind in trans with a small RING finger protein, such as ROC1 (also known as Rbx1 and Hrt1) by the cullins, to recruit and activate an E2 (Jackson et al. 2000;Petroski and Deshaies 2005).A remarkable aspect of cullin E3 ligases is that each cullin can assemble into many distinct cullin-RING-dependent ligases (CRLs) by interacting with a conserved motif present in multiple proteins (Petroski and Deshaies 2005). To recruit specific substrates, CUL1 utilizes an N-terminal domain to bind with a linker protein, SKP1 (Feldman et al. 1997;Skowyra et al. 1997;, which does not interact with other cullins (Michel and Xiong 1998). SKP1 uses a separate domain to bind with a conserved protein motif, the F box, which, via its additional protein-protein interaction modules, recruits various substrates, often phosphorylated, to the CUL1-ROC1 catalytic core. To bring specific substrates to CUL2-and CUL5-dependent ligases, a heterodimeric linker complex containing elongins B and C binds simultaneously to an analogous N-terminal domain in CUL2 and CUL5 and to two similar protein motifs, the VHL box and SOCS box. VHL and SOCS proteins, via their additional protein-protein interaction modules, target various substrates differentially to the CUL2-ROC1 or CUL5-ROC2 catalytic cores (Kamura et al. 1998(Kamura et al. , 2001(Kamura et al. , 2004Stebbins et al. 1999;Zhang et al. 1999). Omitting a linker, CUL3 utilizes its N-terminal domain to bind to proteins with a conserved 100-residue protein motif known as a BTB domain, which, via additional protein-protein interaction domains, then target various substrates to the CUL3-ROC1 catalytic core (Furukawa et al. 2003;Geyer et al. 2003;Pintard et al. 2003;Xu et al. 2003). The presence of multiple substrate receptors-...

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.DOI: http://dx.doi.org/10.7554/eLife.00726.001

Data from patients with coronavirus disease 2019 (COVID-19) are essential for guiding clinical decision making, for furthering the understanding of this viral disease, and for diagnostic modelling. Here, we describe an open resource containing data from 1,521 patients with pneumonia (including COVID-19 pneumonia) consisting of chest computed tomography (CT) images, 130 clinical features (from a range of biochemical and cellular analyses of blood and urine samples) and laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clinical status. We show the utility of the database for prediction of COVID-19 morbidity and mortality outcomes using a deep learning algorithm trained with data from 1,170 patients and 19,685 manually labelled CT slices. In an independent validation cohort of 351 patients, the algorithm discriminated between negative, mild and severe cases with areas under the receiver operating characteristic curve of 0.944, 0.860 and 0.884, respectively. The open database may have further uses in the diagnosis and management of patients with COVID-19.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2’-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2’-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2’-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2–3 and 3–4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35–400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

Recent reports have suggested that the gut microbiota is involved in the progression of colorectal cancer (CRC). The composition of gut microbiota in CRC precursors has not been adequately described. To characterize the structure of adherent microbiota in this disease, we conducted pyrosequencing-based analysis of 16S rRNA genes to determine the bacterial profile of normal colons (healthy controls) and colorectal adenomas (CRC precursors). Adenoma mucosal biopsy samples and adjacent normal colonic mucosa from 31 patients with adenomas and 20 healthy volunteers were profiled using the Illumina MiSeq platform. Principal coordinate analysis (PCoA) showed structural segregation between colorectal adenomatous tissue and control tissue. Alpha diversity estimations revealed higher microbiota diversity in samples from patients with adenomas. Taxonomic analysis illustrated that abundance of eight phyla (Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Cyanobacteria, Candidate-division TM7, and Tenericutes) was significantly different. In addition, Lactococcus and Pseudomonas were enriched in preneoplastic tissue, whereas Enterococcus, Bacillus, and Solibacillus were reduced. However, both PCoA and cluster tree analyses showed similar microbiota structure between adenomatous and adjacent non-adenoma tissues. These present findings provide preliminary experimental evidence supporting that colorectal preneoplastic lesion may be the most important factor leading to alterations in bacterial community composition.

Many different signaling pathways share common components but nevertheless invoke distinct physiological responses. In yeast, the adaptor protein Ste50 functions in multiple mitogen-activated protein (MAP) kinase pathways, each with unique dynamical and developmental properties. Although Kss1 activity is sustained and promotes invasive growth, Hog1 activity is transient and promotes cell adaptation to osmotic stress. Here we show that osmotic stress activates Kss1 as well as Hog1. We show further that Hog1 phosphorylates Ste50 and that phosphorylation of Ste50 limits the duration of Kss1 activation and prevents invasive growth under high osmolarity growth conditions. Thus feedback regulation of a shared component can restrict the activity of a competing MAP kinase to ensure signal fidelity.All living organisms respond to specific external cues and initiate the appropriate developmental or metabolic responses. In many cases, extracellular stimuli lead to activation of MAP 2 kinases, which in turn initiate distinct, and often mutually exclusive, cellular behaviors including cell growth, movement, differentiation, and death. Therefore the regulation and coordination of multiple kinases are essential for the cell to respond appropriately to a changing environment (1). However, the underlying mechanisms ensuring pathway fidelity are not well understood.The MAP kinases in yeast Saccharomyces cerevisiae provide a powerful model to study the mechanisms of signal specificity. Two different MAP kinases, Fus3 and Kss1, function in the mating-response pathway leading to fusion of haploid a-and ␣-type cells. In this case, pheromone stimulation leads to activation of a protein kinase cascade that includes Ste20, Ste11, Ste7, and ultimately Fus3 and Kss1 (2).Nutrient-poor conditions lead to activation of the same kinase components, with the exception of Fus3 (3, 4). In this alternate developmental pathway, the cells exhibit altered budding, formation of long branching filaments, as well as increased adherence and invasion of the substratum. A third pathway leads to activation of the high osmolarity glycerol (HOG) response kinase Hog1 (5, 6). The HOG response is initiated by stimulation of two putative osmosensing proteins, Sln1 and Sho1 (7,8). Sln1 activates two partially redundant kinases, Ssk2 and Ssk22, which then activate the MAP kinase kinase Pbs2 and ultimately Hog1. Sho1 recruits a distinct kinase Ste11 to activate Pbs2 and Hog1. In either case, Hog1 induces production of glycerol that serves to balance intracellular osmotic pressure with the external environment and thereby enables cell survival (9 -11).Thus Ste11 is required for signaling by Sho1 as well as by mating pheromones. Another shared component is Ste50, which forms a stable complex with Ste11 (12-17). Although the Sho1 branch of the Hog1 pathway shares components with the Fus3 and Kss1 pathways, osmotic stimulation does not normally induce mating or invasive growth. If Hog1 expression or catalytic activity is abrogated, however, osmotic stimulation leads to ...