The undernourished neonatal mouse metabolome reveals evidence of liver and biliary dysfunction, inflammation, and oxidative stress.

Undernutrition contributes to half of all childhood deaths under the age of 5 y, and confers upon survivors a life-long predisposition to obesity, type 2 diabetes, and cardiovascular disease. Mechanisms underlying the link between early nutrient deprivation and noncommunicable diseases are unknown. Using outbred CD1 neonatal mice, we measured metabolic profile differences between conventionally reared mice given unrestricted access to nursing, the control group, and undernourished mice subjected to protein-calorie deprivation through timed separation from lactating mothers. After 11 d of undernutrition, urine, plasma, liver, ileal fluid, cecal fluid, and stool were harvested from 8 pools of 4 neonatal mice per group. The metabolome was identified using a multiplatform mass spectrometry-based approach, and random forest metrics were used to identify the most important metabolites that distinguished the undernourished from the control group. Our data reveal striking metabolic changes in undernourished mice consistent with the known mammalian response to starvation, including evidence of muscle and fat catabolism and increased reliance on the tricarboxylic acid cycle for energy. However, we also revealed evidence of liver and biliary injury, anomalies in bile acid metabolism, oxidative stress and inflammation, accelerated heme breakdown, and altered regulation of DNA methylation. Among the metabolites that most strongly distinguished the 2 groups were 2-hydroxyisobutyrate, increased 3-fold in plasma of undernourished mice (P = 2.19 × 10(-11)); urobilinogen, increased 11-fold in urine of undernourished mice (P = 4.22 × 10(-7)); deoxycholate, decreased 94% in stool of undernourished mice (P = 3.0 × 10(-4)); and 12 different products of the enzyme γ-glutamyltransferase, increased in all 6 compartments of undernourished mice. This model of the undernourished neonatal metabolome illustrates the wide range of pathways disrupted by undernutrition in early development, and suggests mechanistic links between early starvation and persistent metabolic diseases.

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation.

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.

Caloric Restriction Alters Postprandial Responses of Essential Brain Metabolites in Young Adult Mice.

Caloric restriction (CR) has been shown to extend longevity and protect brain function in aging. However, the effects of CR in young adult mice remain largely unexplored. In addition to the fundamental, long-term changes, recent studies demonstrate that CR has a significant impact on transient, postprandial metabolic flexibility and turnover compared to control groups. The goal of this study was to identify the brain metabolic changes at a transient (2 h) and steady (6 h) postprandial state in young mice (5-6 months of age) fed with CR or ad libitum (AL; free eating). Using metabolomics profiling, we show that CR mice had significantly higher levels of neurotransmitters (e.g., glutamate, N-acetylglutamate), neuronal integrity markers (e.g., NAA and NAAG), essential fatty acids (e.g., DHA and DPA), and biochemicals associated carnitine metabolism (related to reduced oxidative stress and inflammation) in the cerebral cortex and hippocampus at 2-h. These biochemicals remained at high levels at the 6-h postprandial time-point. The AL mice did not show the similar increases in essential fatty acid and carnitine metabolism until the 6-h time-point, and failed to show increases in neurotransmitters and neuronal integrity markers at any time-point. On the other hand, metabolites related to glucose utilization-glycolysis and pentose phosphate pathway (PPP)-were low in the CR mice throughout the 6-h period and significantly increased at the 6-h time-point in the AL mice. Our findings suggest that CR induces distinct postprandial responses in metabolites that are essential to maintain brain functions. CR mice produced higher levels of essential brain metabolites in a shorter period after a meal and sustained the levels for an extended period, while maintaining a lower level of glucose utilization. These early brain metabolism changes in the CR mice might play a critical role for neuroprotection in aging. Understanding the interplay between dietary intervention and postprandial metabolic responses from an early age may have profound implications for impeding brain aging and reducing risk for neurodegenerative disorders.

Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin.

The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here, we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisting of 12 residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the alpha 3 helix and the central beta-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76(Ub) carboxylate group either by a peptide or isopeptide bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as "sensors" of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes.

Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint.

The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial.

Metabolic effects of acute thiamine depletion are reversed by rapamycin in breast and leukemia cells.

Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the thiaminase-mediated suppression of the TDE transketolase. Interestingly, thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblot analyses showed corresponding differences in expression of BCKDH pathway enzymes, and partial protection of thiaminase growth inhibition by gabapentin indicated that BCKDH inhibition may be a mechanism of thiaminase-mediated toxicity. Surprisingly, most of thiaminase-mediated metabolomic effects were also reversed by rapamycin. Thus, these studies demonstrate that acute intracellular thiamine depletion by recombinant thiaminase results in metabolic changes in thiamine-dependent metabolism, and demonstrate a previously unrecognized role of mTOR signaling in the regulation of thiamine-dependent metabolism.

Genomic instability in cancer.

One of the fundamental challenges facing the cell is to accurately copy its genetic material to daughter cells. When this process goes awry, genomic instability ensues in which genetic alterations ranging from nucleotide changes to chromosomal translocations and aneuploidy occur. Organisms have developed multiple mechanisms that can be classified into two major classes to ensure the fidelity of DNA replication. The first class includes mechanisms that prevent premature initiation of DNA replication and ensure that the genome is fully replicated once and only once during each division cycle. These include cyclin-dependent kinase (CDK)-dependent mechanisms and CDK-independent mechanisms. Although CDK-dependent mechanisms are largely conserved in eukaryotes, higher eukaryotes have evolved additional mechanisms that seem to play a larger role in preventing aberrant DNA replication and genome instability. The second class ensures that cells are able to respond to various cues that continuously threaten the integrity of the genome by initiating DNA-damage-dependent "checkpoints" and coordinating DNA damage repair mechanisms. Defects in the ability to safeguard against aberrant DNA replication and to respond to DNA damage contribute to genomic instability and the development of human malignancy. In this article, we summarize our current knowledge of how genomic instability arises, with a particular emphasis on how the DNA replication process can give rise to such instability.

Exposure of clinical MRSA heterogeneous strains to ß-lactams redirects metabolism to optimize energy production through the TCA cycle.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a ß-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to ß-lactam (HeR) in which only a small portion (≤ 0.1%) of the population expresses resistance to oxacillin (OXA) ≥ 10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a ß-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of ß-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with ß-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished ß-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to ß-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of ß-lactam antibiotics.

DNA replication: mammalian Treslin-TopBP1 interaction mirrors yeast Sld3-Dpb11.

There are many parallels between DNA replication in yeast and humans. Now, two recent studies extend this relationship by dissecting key conserved interactions necessary for initiation of the replisome.

Nuclear scaffold attachment sites within ENCODE regions associate with actively transcribed genes.

The human genome must be packaged and organized in a functional manner for the regulation of DNA replication and transcription. The nuclear scaffold/matrix, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we identified 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate extraction of HeLa nuclei across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites mapped predominately near expressed genes and localized near transcription start sites and the ends of genes but not to boundary elements. In addition, these regions were enriched for RNA polymerase II and transcription factor binding sites and were located in early replicating regions of the genome. We believe these sites correspond to genome-interactions mediated by transcription factors and transcriptional machinery immobilized on a nuclear substructure.

Molecular dissection of the checkpoint kinase Hsl1p.

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.

Nucleocytoplasmic trafficking of G2/M regulators in yeast.

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

The DNA-compacting protein DCP68 from soybean chloroplasts is ferredoxin:sulfite reductase and co-localizes with the organellar nucleoid.

The multiple copies of the chloroplast genome (plastome) are condensed and organized into nucleoids by a set of proteins. One of these, the DNA-binding protein DCP68 from soybean, has previously been shown to compact DNA and to inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is the siroheme protein sulfite reductase, a ferredoxin-dependent enzyme that participates in sulfur assimilation for cysteine and methionine biosynthesis. The in vivo association of this protein with chloroplast nucleoids was confirmed by immuno-colocalization with antibodies against sulfite reductase from Arabidopsis thaliana. These results suggest that DCP68 is a bifunctional chloroplast protein that participates in reductive sulfur assimilation and plays a role in organellar nucleoid organization. The fact that dephosphorylation by alkaline phosphatase affects the binding of purified DCP68 to DNA in vitro might be indicative of the way the interaction of the protein with the nucleoid is regulated in vivo.