Insomnia is a type of sleep disorder which is associated with various diseases’ development and progression, such as obesity, type II diabetes and cardiovascular diseases. Recent investigation of the gut-brain axis enhances our understanding of the role of the gut microbiota in brain-related diseases. However, whether the gut microbiota is associated with insomnia remains unknown. In the present investigation, leveraging the 16S rDNA amplicon sequencing of V3-V4 region and the novel bioinformatic analysis, it was demonstrated that between insomnia and healthy populations, the composition, diversity and metabolic function of the gut microbiota are significantly changed. Other than these, redundancy analysis, co-occurrence analysis and PICRUSt underpin the gut taxa composition, signaling pathways, and metabolic functions perturbed by insomnia disorder. Moreover, random forest together with cross-validation identified two signature bacteria, which could be used to distinguish the insomnia patients from the healthy population. Furthermore, based on the relative abundance and clinical sleep parameter, we constructed a prediction model utilizing artificial neural network (ANN) for auxiliary diagnosis of insomnia disorder. Overall, the aforementioned study provides a comprehensive understanding of the link between the gut microbiota and insomnia disorder.

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of ␣-subunit loop two (␣2) beneath a disulfide "latched" strand of the ␤-subunit known as the "seatbelt." This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free ␤-subunit. We refer to this disulfide as the "tensor" because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin ␤-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the ␤-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the ␤-subunit cystine knot and the tensor loop with backbone atoms in loop ␣2, a process that causes the glycosylated end of loop ␣2 to be threaded between the seatbelt and the ␤-subunit core. Contacts between the tensor loop and loop ␣2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined ␤-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.The glycoprotein hormones are heterodimers of two cystine knot proteins (1-3) in which a glycosylated loop of one subunit (loop ␣2) 1 is surrounded by a strand of the other "like a seatbelt" (1). This topology raises questions as to how these heterodimers might be assembled. We have found that the human glycoprotein hormone subunits combine by a process in which the glycosylated end of loop ␣2 is threaded beneath the seatbelt while it is latched (22). Although the hCG heterodimer can be assembled by a mechanism in which the seatbelt is wrapped around loop ␣2 after the subunits dock (4, 5), this appears to be a minor pathway that can be used to form some hormone analogs that are unable to latch their seatbelts to ␤-subunit loop 1. This "salvage" pathway may have had a role in the evolution of glycoprotein hormones in some teleost fish (23).Purified glycoprotein hormone subunits have long been known to recombine slowly in vitro in oxidizing conditions (6), a phenomenon that occurs while all the disulfides in both subunits remain intact (7). This showed that assembly can occur by a mechanism in which the glycosylated end of loop ␣2 is threaded beneath the seatbelt. hCG assembly is accelerated substantially by protein-disulfide isomerase (8) and low concentrations of reducing agents, however (7). Furthermore, ␤-mercaptoethanol-catalyzed assembly is blocked by agents that react with thiols, e.g. iodo...

Studies described here were initiated to develop a model of glycoprotein hormone receptor structure and function. We found that the region that links the lutropin receptor leucine-rich repeat domain (LRD) to its transmembrane domain (TMD) has substantial roles in ligand binding and signaling, hence we term it the signaling specificity domain (SSD). Theoretical considerations indicated the short SSDs in marmoset lutropin and salmon follitropin receptors have KH domain folds. We assembled models of lutropin, follitropin, and thyrotropin receptors by aligning models of their LRD, TMD, and shortened SSD in a manner that explains how substitutions in follitropin and thyrotropin receptors distant from their apparent ligand binding sites enable them to recognize lutropins. In these models, the SSD is parallel to the concave surface of the LRD and makes extensive contacts with TMD outer loops 1 and 2. The LRD appears to contact TMD outer loop 3 and a few residues in helices 1, 5, 6, and 7. We propose that signaling results from contacts of the ligands with the SSD and LRD that alter the LRD, which then moves TMD helices 6 and 7. The positions of the LRD and SSD support the notion that the receptor can be activated by hormones that dock with these domains in either of two different orientations. This would account for the abilities of some ligands and ligand chimeras to bind multiple receptors and for some receptors to bind multiple ligands. This property of the receptor may have contributed significantly to ligand-receptor co-evolution.

The microbiome has af undamental impact on the human hostsp hysiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes.M ass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed an ew chemical biology tool using chemoselective modification to overcome analytical limitations.T wo isotopic probes allowf or the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery.Weanticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain ab etter understanding of microbial interactions with the human host and disease development.

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the ␣-subunit straddles a hole in the ␤-subunit. This hole is formed when a cysteine at the end of a ␤-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the ␤-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the ␣-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the ␣-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the ␣-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide crosslinked human choriogonadotropin analogs that contain an additional ␣-subunit cysteine, but only if the normal ␤-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of ␣-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.The glycoprotein hormones are essential for vertebrate reproduction and thyroid function. Each of the four human hormones is composed of non-covalently bound ␣-and ␤-subunits that are divided into three large loops (␣1, ␣2, ␣3; ␤1, ␤2, ␤3) 1 by a cystine knot (1-3). The ␤-subunit also contains 20 additional residues that form a strand commonly known as the "seatbelt" because of its topology and its role in stabilizing the heterodimer (1). The seatbelt begins at the ␤-subunit cystine knot and its carboxyl-terminal cysteine is "latched" by a disulfide to a cysteine in loop ␤1. This creates a hole in the ␤-subunit that is bordered on one side by the core of the ␤-subunit and on the other side by the seatbelt. The ␣-subunit straddles this hole such that the glycosylated end of loop ␣2 must pass beneath the seatbelt through the ␤-subunit hole for the heterodimer to dissociate. This contributes to the stability of the heterodimer, which dissociates at low pH or in high concentrations of urea (4), but not in the presence of ionic detergents such as 0.1% sodium dodecyl sulfate. If the seatbelt were to be latched before the subunits combine, the glycosylated end of loop ␣2 would also need to pass through the ␤-subunit during heterodimer assembly. This would impede assembly, a notion supported by the finding that removal of this oligosaccharide accelerates assembly in vitro substantially (5), a process that occurs by a threading m...

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their ␤-subunits that is wrapped around ␣-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the ␤-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the ␣-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of ␣-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10 -20% the activity of hCG. Attachment of the seatbelt to ␣-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the ␤-subunit has folded completely and support the notions that the carboxylterminal end of the seatbelt, portions of ␣-subunit loop 2, and the end of the ␣-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.The heterodimeric placental glycoprotein hormone hCG 1 binds LHR and stimulates ovarian steroid synthesis during early pregnancy (1). The structure of hCG is known (2, 3), but the structures of the LHR and the hormone receptor complex remain to be elucidated. Each hCG subunit is divided into three elongated loops by cystine knots (2, 3), and the heterodimer is stabilized by a part of the ␤-subunit termed the "seatbelt" (2) that is wrapped around ␣-subunit loop 2. The composition of the seatbelt determines the receptor binding specificity of hCG (4 -6), but it is not known if this portion of the hormone contacts the receptor. The LHR is a G protein-coupled receptor that contains a large extracellular domain that binds hCG with high affinity and specificity (7). Based on its leucine-rich repeat motif (7), the LHR extracellular domain is usually presumed to be horseshoe-shaped, similar to ribonuclease inhibitor (8).The surfaces of hCG most likely to contact the LHR remain debated. The carboxyl-terminal end of the ␣-subunit, which is adjacent to a portion of the seatbelt in the heterodimer (2, 3), has been found to influence the affinity of all the glycoprotein hormones for their receptors (1, 9) and was proposed to be a receptor contact more than 25 years ago (1). Based partially on this observation, Jiang et al. (10) suggested that the long axis of hCG docks with the concave surface of a horseshoe-shaped extracellular domain. In their view, the hormone is perpendicular to the extracellular domain of the receptor such that the ␣-subunit carboxyl-terminal end, parts of ␤-subunit loop 2, and the seatbelt...

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000 -10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH ␣-or ␤-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine ␣-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its ␣-subunit and ␤-subunit residue Leu 95 . Leu 95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human ␣-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.The gonadotropins human lutropin (hLH), 1 human chorionic gonadotropin (hCG), and human follitropin (hFSH) are essential for reproduction and have been used for many years to enhance human fertility. Development of clinically useful agonist and antagonist analogs would be facilitated by knowledge of how these ligands interacted with their receptors. Two radically different models of gonadotropin-receptor interaction have been proposed (1, 2) based on the crystal structures of hCG (3, 4) and ribonuclease inhibitor (5, 6), a protein containing a leucine-rich repeat motif thought to be similar to those in the glycoprotein hormone receptors. These models could be readily distinguished if the portions of the hormone that contacted their receptors were known.Like other glycoprotein hormones, the gonadotropins are heterodimers that contain a conserved ␣-subunit and a hormone-specific ␤-subunit (7). Each subunit is divided into three large loops by a cysteine knot (3, 4), and the heterodimer is stabilized by a portion of the ␤-subunit termed the "seat belt" (3) that is wrapped around ␣-subunit loop 2. Based on the activities of chemically and enzymatically modified hormones (summarized by Pierce and Parsons (7)), synthetic hormone fragments (8 -12), and analogs prepared by site-directed mutagenesis (13-19), residues throughout both hormone subunits have been suggested to participate in essential high affinity hormone receptor contacts. Surprisingly, some hCG residues proposed to contact LH receptors are in regions recognized by monoclonal antibodies that bind to hCG-receptor complexes (1,20). Others thought to be essential for receptor contacts can be replaced without disrupting receptor binding. For example, replacing hCG seat belt residues 101-109 with their hFSH counterparts ...

The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treat-ing infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.9 Angstroms resolution, but this does not explain several observations made with cell-surface G protein receptors and may reflect the manner in which FSH binds a short alternate spliced receptor form. We discuss observations that must be explained by any model of the cell-surface G protein-coupled glycoprotein hormone receptors and suggest structures for these receptors that satisfy these requirements. Glycoprotein hormones appear to contact two distinct sites in the extracellular domains of their receptors, not just the leucine-rich repeat domain. These dual contacts contribute to ligand binding specificity and appear to be essential for signal transduction. As outlined in this minireview, differences in the manners in which these ligands contact their receptors explain why some ligands and ligand analogs interact with more than one class of receptor and why some receptors and receptor analogs bind more than one ligand. The unique manner in which these ligands appear to interact with their receptors may have facilitated hormone and receptor co-evolution during early vertebrate speciation.