Role of gut microbiota in sex- and diet-dependent metabolic disorders that lead to early mortality of androgen receptor-deficient male mice.

The gut microbiota is involved in metabolic disorders induced by androgen deficiency after sexual maturation in males (late-onset hypogonadism). However, its role in the energy metabolism of congenital androgen deficiency (e.g., androgen-insensitive syndrome) remains elusive. Here, we examined the link between the gut microbiota and metabolic disease symptoms in androgen receptor knockout (ARKO) mouse by administering high-fat diet (HFD) and/or antibiotics. HFD-fed male, but not standard diet-fed male or HFD-fed female, ARKO mice exhibited increased feed efficiency, obesity with increased visceral adipocyte mass and hypertrophy, hepatic steatosis, glucose intolerance, insulin resistance, and loss of thigh muscle. In contrast, subcutaneous fat mass accumulated in ARKO mice irrespective of the diet and sex. Notably, all HFD-dependent metabolic disorders observed in ARKO males were abolished after antibiotics administration. The ratios of fecal weight-to-food weight and cecum weight-to-body weight were specifically reduced by ARKO in HFD-fed males. 16S rRNA sequencing of fecal microbiota from HFD-fed male mice revealed differences in microbiota composition between control and ARKO mice. Several genera or species (e.g., Turicibacter and Lactobacillus reuteri, respectively) were enriched in ARKO mice, and antibiotics treatment spoiled the changes. Furthermore, the life span of HFD-fed ARKO males was shorter than that of control mice, indicating that androgen deficiency causes metabolic dysfunctions leading to early death. These findings also suggest that AR signaling plays a role in the prevention of metabolic dysfunctions, presumably by influencing the gut microbiome, and improve our understanding of health consequences in subjects with hypogonadism and androgen insensitivity.

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat.

Purification, crystallization and X-ray diffraction study of basic 7S globulin from soybean.

Basic 7S globulin (Bg7S) is expressed by soybeans in response to biotic or abiotic stress. Bg7S is capable of binding to a 4 kDa protein which is supposedly involved in cell proliferation. Bg7S is widely found not only in legumes, but also in other plants; however, its function is still unclear. Here, Bg7S was successfully crystallized. Orthorhombic and monoclinic crystals of Bg7S were obtained under different conditions and belonged to space groups P2(1)2(1)2, with unit-cell parameters a=111.9, b=130.1, c=287.8 Å, and P2(1), with unit-cell parameters a=85.3, b=137.6, c=162.1 Å, ß=91.2°, respectively.

Relationship between gut environment, feces-to-food ratio, and androgen deficiency-induced metabolic disorders.

Androgen action generates sex-related differences that include changes in the gut microbiota composition. Hypoandrogenism and hyperandrogenism in males and females, respectively, are associated with the prevalence of metabolic disorders. Our recent work showed that male androgen receptor knockout (ARKO) mice developed high-fat diet (HFD)-dependent sarcopenic abdominal obesity, hyperglycemia, and hepatic steatosis, leading to early death. The ARKO mice also exhibited alterations in intestinal microbiota but did not experience metabolic abnormalities when administered with antibiotics. Here, we show that time-dependent changes in feed efficiency (ratio of body weight gain to food intake) and weight of dried feces-to-food ratio could be good markers for changes in gut microbiota. Turicibacter spp., Lactobacillus spp., and L. reuteri increased in the gut in both HFD-fed ARKO and castrated mice having metabolic abnormalities. HFD-fed ARKO mice showed increased plasma levels of aspartate, but not alanine, aminotransferase. Changes in the gut microbiome appear to provoke androgen deficiency-induced metabolic diseases, leading to early mortality.

Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Hypogonadism alters cecal and fecal microbiota in male mice.

Low testosterone levels increase the risk for cardiovascular disease in men and lead to shorter life spans. Our recent study showed that androgen deprivation via castration altered fecal microbiota and exacerbated risk factors for cardiovascular disease, including obesity, impaired fasting glucose, excess hepatic triglyceride accumulation, and thigh muscle weight loss only in high-fat diet (HFD)-fed male mice. However, when mice were administered antibiotics that disrupted the gut microbiota, castration did not increase cardiovascular risks or decrease the ratio of dried feces to food intake. Here, we show that changes in cecal microbiota (e.g., an increased Firmicutes/Bacteroidetes ratio and number of Lactobacillus species) were consistent with changes in feces and that there was a decreased cecal content secondary to castration in HFD mice. Castration increased rectal body temperature and plasma adiponectin, irrespective of diet. Changes in the gut microbiome may provide novel insight into hypogonadism-induced cardiovascular diseases.

The analysis of N-glycolylneuraminic acid(NeuGc) of hepatoma tissue and K562 cell ferritins using HPLC and mass spectrometry.

Ferritin is an iron-storage protein and its serum level is known to increase in the patient of with inflammation and malignant tumor. To further elucidate the difference between ferritins from normal human liver tissue and that of cancer cells, their sialic acids were analyzed. The Western blot analysis and the cytochemical staining using anti-NeuGc antiserum indicated that ferritins from the human hepatocarcinoma tissue and malignant K562 cells contain NeuGc, but that from the normal liver does not. The result was also confirmed by HPLC analysis and MALDI-TOF/MS analysis of sialic acids which were derivatized by the DMB method. It was also shown that the sialic acid content in hepatocarcinoma ferritin was much higher than that in the normal liver ferritin. These results suggest that normal and cancerous liver ferritins are qualitatively and quantitatively different in sialylation. In addition, K562 cells were shown to express NeuGc even if the cells were cultured in serum-free media which lack NeuGc. This is of interest from the current concept that expression of NeuGc in human cells is due to uptake and utilization of exogenous NeuGc.

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean.

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.

Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein.

Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation. As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction. However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood. We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein. Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein. To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed. The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants. Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide. Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein.

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.

A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes.

Previously, we isolated a 4-kDa peptide capable of binding to a 43-kDa receptor-like protein and stimulating protein kinase activity of the 43-kDa protein in soybean. Both of them were found to localize in the plasma membranes and cell walls. Here, we report the physiological effects of 4-kDa peptide expressed transiently in the cultured carrot and bird's-foot trefoil cells transfected with pBI 121 plasmid containing the 4-kDa peptide gene. At early developmental stage, the transgenic callus grew rapidly compared to the wild callus in both species. Cell proliferation of in vitro cultured nonembryogenic carrot callus was apparently affected with the 4-kDa peptide in the medium. Complementary DNAs encoding the 4-kDa peptide from mung bean and azuki bean were cloned by PCR and sequenced. The amino-acid sequences deduced from the nucleotide sequences are homologous among legume species, particularly, the sites of cysteine residues are highly conserved. This conserved sequence reflects the importance of intradisulfide bonds required for the 4-kDa peptide to perform its function. Three dimensional structure of the 4-kDa peptide determined by NMR spectroscopy suggests that this peptide is a T-knot scaffold containing three beta-strands, and the specific binding activity to the 43-kDa protein and stimulatory effect on the protein phosphorylation could be attributed to the spatial arrangements of hydrophobic residues at the solvent-exposed surface of two-stranded beta-sheet of 4-kDa peptide. The importance of these residues for the 4-kDa peptide to bind to the 43-kDa protein was indicated by site-directed mutagenesis. These results suggest that the 4-kDa peptide is a hormone-like peptide and the 43-kDa protein is involved in cellular signal transduction of the peptide.