Gender-Specific Metabolomic Profiling of Obesity in Leptin-Deficient ob/ob Mice by 1H NMR Spectroscopy

Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient ob/ob mice based on the gender. Metabolomic analyses of urine and serum from ob/ob mice compared with those from C57BL/6J lean mice, based on the ¹H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice. We also identified 48 urine and 22 serum metabolites that were statistically significantly altered in obese mice compared to lean controls. These metabolites are involved in amino acid metabolism (leucine, alanine, ariginine, lysine, and methionine), tricarbocylic acid cycle and glucose metabolism (pyruvate, citrate, glycolate, acetoacetate, and acetone), lipid metabolism (cholesterol and carnitine), creatine metabolism (creatine and creatinine), and gut-microbiome-derived metabolism (choline, TMAO, hippurate, p-cresol, isobutyrate, 2-hydroxyisobutyrate, methylamine, and trigonelline). Notably, our metabolomic studies showed distinct gender variations. The obese male mice metabolism was specifically associated with insulin signaling, whereas the obese female mice metabolism was associated with lipid metabolism. Taken together, our study identifies the biomarker signature for obesity in ob/ob mice and provides biochemical insights into the metabolic alteration in obesity based on gender.     

Candidate target genes for the Saccharomyces cerevisiae transcription factor, Yap2

In Saccharomyces cerevisiae, the Yap family of basic leucine zipper (bZip) proteins contains eight members. The Yap family proteins are implicated in a variety of stress responses; among these proteins, Yap1 acts as a major regulator of oxidative stress responses. However, the functional roles of the remaining Yap family members are poorly understood. To elucidate the function of Yap2, we mined candidate target genes of Yap2 by proteomic analysis. Among the identified genes, FRM2 was previously identified as a target gene of Yap2, which confirmed the validity of our screening method. YNL134C and YDL124W were also identified as candidate Yap2 target genes. These genes were upregulated in strains overexpressing Yap2 and possess Yap2 target sequences in their promoter regions. Furthermore, chromatin immunoprecipitation assays showed that YNL134C and YDL124W have Yap2 binding motif. These data will help to elucidate the functional role of Yap2.     

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.     

Solution conformation of alphaA-conotoxin EIVA,a potent neuromuscular nicotinic acetylcholine receptor antagonist from Conus ermineus

We report the solution three-dimensional structure of an alphaA-conotoxin EIVA determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The alphaA-conotoxin EIVA consists of 30 amino acids representing the largest peptide among the alpha/alphaA-family conotoxins discovered so far and targets the neuromuscular nicotinic acetylcholine receptor with high affinity. alphaA-Conotoxin EIVA consists of three distinct structural domains. The first domain is mainly composed of the Cys3-Cys11-disulfide loop and is structurally ill-defined with a large backbone root mean square deviation of 1.91 A. The second domain formed by residues His12-Hyp21 is extremely well defined with a backbone root mean square deviation of 0.52 A, thus forming a sturdy stem for the entire molecule. The third C-terminal domain formed by residues Hyp22-Gly29 shows an intermediate structural order having a backbone root mean square deviation of 1.04 A. A structurally ill-defined N-terminal first loop domain connected to a rigid central molecular stem seems to be the general structural feature of the alphaA-conotoxin subfamily. A detailed structural comparison between alphaA-conotoxin EIVA and alphaA-conotoxin PIVA suggests that the higher receptor affinity of alphaA-conotoxin EIVA than alphaA-conotoxin PIVA might originate from different steric disposition and charge distribution in the second loop "handle" motif.     

Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-A resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I.5 enzymes in comparison to other microbial lipases. The zinc-coordinated extra domain makes tight interactions with the loop extended from the C terminus of the lid helix, suggesting that the activation of the family I.5 lipases may be regulated by the strength of the interactions. The unusually long lid helix makes strong hydrophobic interactions with its neighbors. The structural information together with previous biochemical observations indicate that the temperature-mediated lid opening is triggered by the thermal dissociation of the hydrophobic interactions.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

Pre-structured motifs in the natively unstructured preS1 surface antigen of hepatitis B virus

The preS1 surface antigen of hepatitis B virus (HBV) is known to play an important role in the initial attachment of HBV to hepatocytes. We have characterized structural features of the full-length preS1 using heteronuclear NMR methods and discovered that this 119-residue protein is inherently unstructured without a unique tertiary structure under a nondenaturing condition. Yet, combination of various NMR parameters shows that the preS1 contains "pre-structured" domains broadly covering its functional domains. The most prominent domain is formed by residues 27-45 and overlaps with the putative hepatocyte-binding domain (HBD) encompassing residues 21-47, within which two well-defined pre-structured motifs, formed by Pro(32)-Ala(36) and Pro(41)-Phe(45) are found. Additional, somewhat less prominent, pre-structured motifs are also formed by residues 11-18, 22-25, 37-40, and 46-50. Overall results suggest that the preS1 is a natively unstructured protein (NUP) whose N-terminal 50 residues, populated with multiple pre-structured motifs, contribute critically to hepatocyte binding.     

The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits

The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome.