Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response.

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.

Postweaning Iron Deficiency in Male Rats Leads to Long-Term Hyperactivity and Decreased Reelin Gene Expression in the Nucleus Accumbens.

BACKGROUND: Epidemiological research indicates that iron deficiency (ID) in infancy correlates with long-term cognitive impairment and behavioral disturbances, despite therapy. However, the mechanisms underlying these effects are unknown. OBJECTIVE: We investigated how ID affected postweaning behavior and monoamine concentration in rat brains to determine whether ID during the juvenile period affected gene expression and synapse formation in the prefrontal cortex (PFC) and nucleus accumbens (NAcc). METHODS: Fischer 344/Jcl postweaning male rats aged 21-39 d were fed low-iron diets (0.35 mg/kg iron; ID group) or standard AIN-93 G diets [3.5 mg/kg iron; control (CN) group]. After day 39, all rats were fed the iron-adequate diet. The locomotor activity was evaluated by the open field and elevated plus maze tests at 8 and 12 wk of age. Monoamine concentrations in the brain were analyzed using HPLC at 9 and 13 wk of age. Comprehensive gene expression analysis was performed in the PFC and NAcc at 13 wk of age. Finally, we investigated synaptic density in the PFC and NAcc by synaptophysin immunostaining. RESULTS: Behavioral tests revealed a significant reduction of the age-related decline in the total distance traveled in ID rats compared with CN rats (P < 0.05), indicating that ID affected hyperactivity, which persisted into adulthood (13 wk of age). At this age, reelin (Reln) mRNA expression (adjusted P < 0.01) decreased and synaptic density (P < 0.01) increased in the NAcc in the ID group. Regarding the mesolimbic pathway, homovanillic acid concentration increased in the NAcc, whereas the dopamine concentration decreased in the ventral midbrain. CONCLUSIONS: Our results suggest that ID during the postweaning period in male rats, despite complete iron repletion following ID, led to long-term hyperactivity via monoamine disturbance in the brain and an alteration in the synaptic plasticity accompanied by downregulation of Reln expression in the NAcc.

Solitary and Synergistic Effects of Different Hydrophilic and Hydrophobic Phospholipid Moieties on Rat Behaviors.

Glycerophospholipids have hydrophobic and hydrophilic moieties. Previous studies suggest that phospholipids with different moieties have different effects on rodent behavior; however, the relationship between chemical structures and behavioral effects remains unclear. To clarify the functions of phospholipid moieties, we injected male rats with phospholipids with different moieties and conducted behavioral tests. Exploratory activity was reduced by phosphatidylethanolamine (PE)(18:0/22:6) but not PE(18:0/18:0) or PE(18:0/20:4). Conversely, exploratory activity was increased by plasmanyl PE(16:0/22:6), which harbors an alkyl-ether linkage, but not by phosphatidylcholine (PC)(16:0/22:6) or plasmanyl PC(16:0/22:6). Docosahexaenoic acid (DHA)(22:6) and an alkyl-ether linkage in PE were thus postulated to be involved in exploratory activity. Anxiety-like behavior was reduced by plasmenyl PC(18:0/20:4), which harbors a vinyl-ether linkage, but not by PC(18:0/20:4) or plasmanyl PC(18:0/20:4), suggesting the anxiolytic effects of vinyl-ether linkage. The activation of social interaction was suppressed by PE(18:0/18:0), PE(18:0/22:6), PC(16:0/22:6), plasmanyl PE(16:0/22:6), and plasmanyl PC(16:0/22:6) but not by PE(18:0/20:4), plasmenyl PE(18:0/20:4), or plasmanyl PC(18:0/22:6). DHA may suppress social interaction, whereas arachidonic acid(20:4) or a combination of alkyl-ether linkage and stearic acid(18:0) may restore social deficits. Our findings indicate the characteristic effects of different phospholipid moieties on rat behavior, and may help to elucidate patterns between chemical structures and their effects.

Excessive folic acid intake combined with undernutrition during gestation alters offspring behavior and brain monoamine profiles.

Dietary folic acid augmentation during gestation reduces neurodevelopmental disorder risk in offspring; however, it is still unclear if excessive maternal folic acid intake can impair brain function in offspring. We examined if excessive folic acid intake throughout gestation altered the behavior of male offspring under poor nutrition during early gestation (E5.5-E11.5). Dams were divided into four groups: control (CON, 2 mg folic acid/kg of food), excessive folic acid fortification (FF, 10 mg folic acid/kg of food), undernutrition (UN, 40% food reduction from E5.5-E11.5), and excessive folic acid fortification plus undernutrition (UN-FF). Excess maternal folic acid fortification induced hyperactivity in the open-field and lower anxiety-like behavior in the elevated plus maze at 9 weeks of age. These behavioral changes were accompanied by reduced dopamine in the prefrontal cortex (PFC), norepinephrine in the amygdala, and 5-hydroxytryptamine (5-HT) in the dorsal midbrain (DM), PFC, and amygdala where 5-HT neurons project from the DM. Furthermore, canonical discriminant analysis, including dopamine and DOPAC concentrations in the PFC, norepinephrine concentrations in the PFC, amygdala, and pons, and 5-HT and 5-HIAA concentrations in the amygdala and DM, correctly classified 73.5% of the offspring in CON, FF, UN, and UN-FF groups. The first discriminant function mainly classified groups based on nutritional status, whereas the second function mainly classified groups based on folic acid intake. Our study suggests that combined transformations of brain monoamine profiles by maternal undernutrition and excess folic acid intake is involved in the behavioral alteration of offsprings.

Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.

Members of the small ubiquitin-like modifier (SUMO) family can be covalently attached to the lysine residue of a target protein through an enzymatic pathway similar to that used in ubiquitin conjugation, and are involved in various cellular events that do not rely on degradative signalling via the proteasome or lysosome. However, little is known about the molecular mechanisms of SUMO-modification-induced protein functional transfer. During DNA mismatch repair, SUMO conjugation of the uracil/thymine DNA glycosylase TDG promotes the release of TDG from the abasic (AP) site created after base excision, and coordinates its transfer to AP endonuclease 1, which catalyses the next step in the repair pathway. Here we report the crystal structure of the central region of human TDG conjugated to SUMO-1 at 2.1 A resolution. The structure reveals a helix protruding from the protein surface, which presumably interferes with the product DNA and thus promotes the dissociation of TDG from the DNA molecule. This helix is formed by covalent and non-covalent contacts between TDG and SUMO-1. The non-covalent contacts are also essential for release from the product DNA, as verified by mutagenesis.

Global pattern of interkinetic nuclear migration in tracheoesophageal epithelia of the mouse embryo: Interorgan and intraorgan regional differences.

Interkinetic nuclear migration (INM) is an apicobasal (AB) polarity-based regulatory mechanism of proliferation/differentiation in epithelial stem/progenitor cells. We previously documented INM in the endoderm-derived tracheal/esophageal epithelia at embryonic day (E) 11.5 and suggested that INM is involved in the development of both organs. We here investigated interorgan (trachea vs esophagus) and intraorgan regional (ventral vs dorsal) differences in the INM mode in the tracheal and esophageal epithelia of the mouse embryo. We also analyzed convergent extension (CE) and planar cell movement (PCM) in the epithelia based on cell distribution. The pregnant C57BL/6J mice were intraperitoneally injected with 5-ethynyl-2'-deoxyuridine at E11.5 and E12.5 and were sacrificed 1, 4, 6, 8, and 12 hours later to obtain the embryos. The distribution of labeled cell nuclei along the AB axis was chronologically analyzed in the total, ventral, and dorsal sides of the epithelia. The percentage distribution of the nuclei population was represented by histogram and the chronological change was analyzed statistically using multidimensional scaling. The interorgan comparison of the INM mode during E11.5-E12.0, but not E12.5-E13.0, showed a significant difference. During E11.5-E12.0 the trachea, but not the esophagus, showed a significant difference between ventral and dorsal sides. During E12.5-E13.0 neither organ showed regional differences. CE appeared to occur in both organs during E11.5-E12.0 while PCM was unclear in both organs. These findings suggest a difference between the trachea and esophagus, and a regional difference in the trachea, not in the esophagus, in the INM mode, which may be related with the later differential organogenesis/histogenesis of these organs.

The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci.

The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.

Correlation between musculoskeletal structure of the hand and primate locomotion: Morphometric and mechanical analysis in prehension using the cross- and triple-ratios.

Biometric ratios of the relative length of the rays in the hand have been analyzed between primate species in the light of their hand function or phylogeny. However, how relative lengths among phalanges are mechanically linked to the grasping function of primates with different locomotor behaviors remains unclear. To clarify this, we calculated cross and triple-ratios, which are related to the torque distribution, and the torque generation mode at different joint angles using the lengths of the phalanges and metacarpal bones in 52 primates belonging to 25 species. The torque exerted on the finger joint and traction force of the flexor tendons necessary for a cylindrical grip and a suspensory hand posture were calculated using the moment arm of flexor tendons measured on magnetic resonance images, and were compared among Hylobates spp., Ateles sp., and Papio hamadryas. Finally, the torques calculated from the model were validated by a mechanical study detecting the force exerted on the phalanx by pulling the digital flexor muscles during suspension in these three species. Canonical discriminant analysis of cross and triple-ratios classified primates almost in accordance with their current classification based on locomotor behavior. The traction force was markedly reduced with flexion of the MCP joint parallel to the torque in brachiating primates; this was notably lower in the terrestrial quadrupedal primates than in the arboreal primates at mild flexion. Our mechanical study supported these features in the torque and traction force generation efficiencies. Our results suggest that suspensory or terrestrial quadrupedal primates have hand structures that can exert more torque at a suspensory posture, or palmigrade and digitigrade locomotion, respectively. Furthermore, our study suggests availability of the cross and triple-ratios as one of the indicators to estimate the hand function from the skeletal structure.

MafG sumoylation is required for active transcriptional repression.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.

Crystal structure of SUMO-3-modified thymine-DNA glycosylase.

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular beta-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM.

Complete Genome Sequences of 12 Species of Stable Defined Moderately Diverse Mouse Microbiota 2.

We report here the complete genome sequences of 12 bacterial species of stable defined moderately diverse mouse microbiota 2 (sDMDMm2) used to colonize germ-free mice with defined microbes. Whole-genome sequencing of these species was performed using the PacBio sequencing platform yielding circularized genome sequences of all 12 species.

The maternal microbiota drives early postnatal innate immune development.

Postnatal colonization of the body with microbes is assumed to be the main stimulus to postnatal immune development. By transiently colonizing pregnant female mice, we show that the maternal microbiota shapes the immune system of the offspring. Gestational colonization increases intestinal group 3 innate lymphoid cells and F4/80(+)CD11c(+) mononuclear cells in the pups. Maternal colonization reprograms intestinal transcriptional profiles of the offspring, including increased expression of genes encoding epithelial antibacterial peptides and metabolism of microbial molecules. Some of these effects are dependent on maternal antibodies that potentially retain microbial molecules and transmit them to the offspring during pregnancy and in milk. Pups born to mothers transiently colonized in pregnancy are better able to avoid inflammatory responses to microbial molecules and penetration of intestinal microbes.

Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway.

Here, we developed a binary vector system that introduces a synthetic SUMO-1 conjugation pathway into Escherichia coli and demonstrated that large amounts of sumoylated Ran GTPase activating protein 1 C-terminal region (RanGAP1-C2), Ran binding protein 2 internal repeat domain, p53 and promyelocytic leukemia were efficiently produced. The sumoylated recombinant RanGAP1-C2 appeared to retain the in vivo properties, since it was specifically sumoylated at lysine 517 as expected from in vivo studies. Our findings indicate the establishment of a biosynthetic route for producing large amounts of sumoylated recombinant proteins that will open up new avenues for studying the biochemical and structural aspects of the SUMO-1 modification pathway.

REG-γ associates with and modulates the abundance of nuclear activation-induced deaminase.

Activation-induced deaminase (AID) acts on the immunoglobulin loci in activated B lymphocytes to initiate antibody gene diversification. The abundance of AID in the nucleus appears tightly regulated, with most nuclear AID being either degraded or exported back to the cytoplasm. To gain insight into the mechanisms regulating nuclear AID, we screened for proteins interacting specifically with it. We found that REG-γ, a protein implicated in ubiquitin- and ATP-independent protein degradation, interacts in high stoichiometry with overexpressed nuclear AID as well as with endogenous AID in B cells. REG-γ deficiency results in increased AID accumulation and increased immunoglobulin class switching. A stable stoichiometric AID-REG-γ complex can be recapitulated in co-transformed bacteria, and REG-γ accelerates proteasomal degradation of AID in in vitro assays. Thus, REG-γ interacts, likely directly, with nuclear AID and modulates the abundance of this antibody-diversifying but potentially oncogenic enzyme.

Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions.

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

Structure of the small ubiquitin-like modifier (SUMO)-interacting motif of MBD1-containing chromatin-associated factor 1 bound to SUMO-3.

Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMO-interacting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel beta-sheet pairing with strand beta2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3.

Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth.

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control.

Involvement of SUMO modification in MBD1- and MCAF1-mediated heterochromatin formation.

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.

In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies.

SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. Interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function.