Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

In mammals, multiple physiological, metabolic, and behavioral processes are subject to circadian rhythms, adapting to changing light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. Disruption of the host circadian clock by deletion of Bmal1, a gene encoding a core molecular clock component, abolished rhythmicity in the fecal microbiota composition in both genders. Bmal1 deletion also induced alterations in bacterial abundances in feces, with differential effects based on sex. Thus, although host behavior, such as time of feeding, is of recognized importance, here we show that sex interacts with the host circadian clock, and they collectively shape the circadian rhythmicity and composition of the fecal microbiota in mice.The composition of intestinal microbiota is influenced by host genetics (1), aging (2), antibiotic exposure (3), lifestyle (4), diet (5), pet ownership (6), and concomitant disease (7, 8). The impact of diet in shaping the composition of the microbiota has been well established in both humans and mice (9, 10). The type of food consumed and also the feeding behavior of the host influence the microbiota. For example, a 24-h fast increases the abundance of Bacteroidetes and reduces that of Firmicutes in mouse cecum, without altering the communal microbial diversity (11). Bacteroidetes are also dominant in the microbiota of the fasted Burmese python, whereas ingestion of a meal shifts the intestinal composition toward Firmicutes (12).The rotation of the earth results in the oscillation of light during the 24-h cycle. Organisms adapted to this cycle by developing a circadian rhythm, an endogenous and entrainable mechanism that times daily events such as feeding, temperature, sleep-wakefulness, hormone secretion, and metabolic homeostasis (13, 14). In mammals, this rhythm is controlled by a master clock that resides in the suprachiasmatic nucleus of the hypothalamus. It responds to the changing light cycle and signals this information to peripheral clocks in most tissues (15). The core mammalian clock is comprised of activators BMAL1 and CLOCK as well as repressors PERIOD (PER) and CRYPTOCHROME (CRY), forming an interlocked regulatory loop (14).Circadian rhythms also exist in fungi and cyanobacteria (16). For example, a pacemaker in cyanobacteria transduces the oscillating daylight signal to regulate gene expression and to time cell division (17, 18). Hence, the synchronization of endogenous circadian rhythms with the environment is crucial for the survival of the bacteria as well as metazoa.Recent studies show that the intestinal microbiota undergo diurnal oscillation under the control of host feeding time, and that ablation of the host molecular clock Per genes causes dysbiosis (19, 20). Here, we report that microbial composition and its oscillation are influenced by the host clock, including the Bmal1-dependent forward limb of the signaling pathway. We also find that rhythmicity is conditioned by the sex of the host, being more pronounced in females than in males.     

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90_N (TRAP1_N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.Heat-shock protein of 90 kDa (Hsp90) is a conserved ATP-dependent molecular chaperone (1–4), which together with heat-shock protein of 70 kDa (Hsp70) (5–7) and a cohort of cochaperones (8–10), promotes the late-stage folding of Hsp90 client proteins (11). It is presumed that almost 400 different proteins, including a majority of signaling and tumor promoting proteins, depend on cytosolic Hsp90 for folding (12). Consequently, the ability to inactivate multiple oncogenic pathways simultaneously has made Hsp90 a major target for drug development (13), with several Hsp90 inhibitors currently undergoing clinical trials (14).Hsp90 chaperones display conformational plasticity in solution (2, 15, 16), with different adenine nucleotides either facilitating or stabilizing distinct Hsp90 dimer conformations (17–19). Interestingly, apo Hsp90 forms a wide-open, V-shaped dimer with the N domains separated by as much as 101 Å (18). This open conformation is markedly distinct from the intertwined, N-terminally closed dimer with ATP bound (20, 21). Because the open-state dimer cannot signal the nucleotide status between neighboring subunits, an intermediate conformation preceding dimer closure must exist, which so far has remained elusive.Apart from cytosolic Hsp90s, Hsp90 homologs are found in the endoplasmic reticulum, chloroplasts, and mitochondria (Fig. S1) (22). The tumor necrosis factor receptor-associated protein 1 (TRAP1) is the mitochondrial Hsp90 paralog, which prevents apoptosis and protects mitochondria against oxidative damage (23–25). TRAP1 is widely expressed in many tumors (24, 26, 27), but not in mitochondria of most normal tissues (24), benign prostatic hyperplasia (26), or highly proliferating, nontransformed cells (27). Notably, it was found that TRAP1 not only promotes neoplastic growth, but also confers tumorigenic potential on nontransformed cells (27), indicating a major role of TRAP1 in tumorigenesis, although TRAP1’s specific function remains poorly understood (28).Open in a separate windowFig. S1.Multiple sequence alignment of the N-terminal nucleotide-binding domain of TRAP1 (mitochondria), HtpG (eubacteria), Hsp90 (cytosolic), and Grp94 (endoplasmic reticulum) from Homo sapiens (Hs), Danio rerio (Dr), E. coli (Ec), and S. cerevisiae (Sc). Residues conserved across sequences of representative members are highlighted in green, and those that are similar are in yellow. Residues not found in the mature protein are shown in lowercase letters. The N strap, ATP lid, and G1 and G2 boxes are marked. Residues that define the G1 and G2 box motifs are in red. Asp158, Phe183, Gln200, and Phe201 are surrounded by a gray box.TRAP1 is a multidomain protein consisting of an N-terminal or N domain (TRAP1_N), a middle domain (TRAP1_M), and a C-terminal or C domain (TRAP1_C), but it lacks the charged linker found in eukaryotic Hsp90 paralogs. Human TRAP1 is preceded by a mitochondrial localization sequence (MLS) of 59 residues that are cleaved off during import (29). The mature form of TRAP1 is a homodimer held together by TRAP1_C, with a second, ATP binding-dependent dimer interface in TRAP1_N. The crystal structures of zebrafish TRAP1 (zTRAP1) and zebrafish and human TRAP1_NM bound to ADPNP were recently reported (21, 30), and are largely consistent with our current understanding of Hsp90 chaperones. In the ATP-bound state, the N-terminal extension (known as the N strap) straddles the N domain of the neighboring subunit, thereby stabilizing the structure of the closed-state dimer (21, 30). The ordered segment of the N strap significantly lengthens the previously observed β-strand swap and may function as a regulatory element that controls TRAP1 function (16, 21). Interestingly, intact zTRAP1-ADPNP crystallized as an asymmetric dimer that could support a sequential ATP hydrolysis mechanism (31, 32); however, no asymmetric nucleotide binding was observed, and no molecular contacts between cis-bound ADPNP and the N domain of the neighboring subunit were seen in TRAP1 (21, 30) and other known Hsp90 structures (18, 20, 33), leaving open the question of how TRAP1 senses and signals the nucleotide-bound status between subunits.Here we present atomic structures of human TRAP1_N (hTRAP1_N) alone and in complex with ADPNP. Unexpectedly, we found that unliganded hTRAP1_N forms a previously unobserved coiled-coil dimer that is distinct from the proposed open-state and closed-state conformations (16, 21, 30). Importantly, intact hTRAP1 forms a similar coiled-coil dimer in solution, but only in the absence of ATP. Our findings show that ATP binding triggers a dramatic change in local structure and displaces the ATP lid, which is bound to the ATP-binding pocket, indicating that TRAP1 normally exists in an autoinhibited state. Strikingly, mutations of conserved residues that impair lid binding stimulate the hTRAP1 ATPase activity in a highly cooperative manner, supporting a previously unknown role of the ATP lid in signaling the cis-bound nucleotide status to the trans subunit, which is compatible with the coiled-coil dimer. Finally, we demonstrate that TRAP1 folding requires ATP and the functional cooperation of the mitochondrial Hsp70 chaperone system, supporting the existence of a mitochondrial Hsp90-Hsp70 supercomplex that may present a new target for drug development.     

CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Light-activated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K⁺) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk^−/− mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K⁺ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go–related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.CRYPTOCHROME (CRY) is a photoreceptor that mediates rapid membrane depolarization and increased spontaneous action potential firing rate in response to blue light in arousal and circadian neurons in Drosophila melanogaster (1, 2). CRY regulates circadian entrainment by targeting circadian clock proteins to proteasomal degradation in response to light (3–6). CRY is expressed in a small subset of central brain circadian, arousal, and photoreceptor neurons in D. melanogaster and other insects, including the large lateral ventral neuron (LNv; l-LNv) subset (1, 2, 7, 8). The l-LNvs are light-activated arousal neurons (1, 2, 9–11), whereas the small lateral ventral neurons (s-LNvs) are critical for circadian function (5, 12). Previous results suggest that light activated arousal is likely attenuated in cry-null mutants. In addition to modulating light-activated firing rate, membrane excitability in the LNv neurons helps maintain circadian rhythms (9, 13, 14), and LNv firing rate is circadian regulated (2, 16).Based on our previous work suggesting that l-LNv electrophysiological light response requires a flavin-specific redox reaction and modulation of membrane K⁺ channels, we investigated the molecular mechanism for CRY phototransduction to determine how light-activated CRY is coupled to rapid membrane electrical changes. Sequence and structural data suggest that the cytoplasmic Kvβs are redox sensors based on a highly conserved aldo-keto-reductase domain (AKR) (17–21). Although no functional role for redox sensing by Kvβ subunits has been established yet in vivo, studies with heterologously expressed WT and mutant Kvβ subunits show that they confer modulatory sensitivity to coexpressed K⁺ channels in response to oxidizing and reducing chemical agents (22–24). Mammals express six Kvβ genes, whereas Drosophila expresses a single Kvβ designated HYPERKINETIC (Hk) (18). We find that the light-activated redox reaction of the flavin adenine dinucleotide (FAD) chromophore in CRY has a distinct phototransduction mechanism that evokes membrane electrical responses via the Kvβ subunit Hk, which we show is a functional redox sensor in vivo.     

A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait

In humans, the connection between sleep and mood has long been recognized, although direct molecular evidence is lacking. We identified two rare variants in the circadian clock gene PERIOD3 (PER3-P415A/H417R) in humans with familial advanced sleep phase accompanied by higher Beck Depression Inventory and seasonality scores. hPER3-P415A/H417R transgenic mice showed an altered circadian period under constant light and exhibited phase shifts of the sleep-wake cycle in a short light period (photoperiod) paradigm. Molecular characterization revealed that the rare variants destabilized PER3 and failed to stabilize PERIOD1/2 proteins, which play critical roles in circadian timing. Although hPER3-P415A/H417R-Tg mice showed a mild depression-like phenotype, Per3 knockout mice demonstrated consistent depression-like behavior, particularly when studied under a short photoperiod, supporting a possible role for PER3 in mood regulation. These findings suggest that PER3 may be a nexus for sleep and mood regulation while fine-tuning these processes to adapt to seasonal changes.In human populations, alterations in circadian timing can result in mood-related problems (1). An example of this is seasonal affective disorder, also known as “winter depression,” which is among the most common mood disorders, with a reported prevalence of 1.5–9%, depending on latitude (2). In addition, shift work has been suggested as a risk factor for major depressive disorder (3), and depression severity correlates with the degree of circadian misalignment (4, 5). A number of genetic variants in core clock genes have been reported as statistically associated with mood disorders, including seasonal affective disorder and major depressive disorder (6–14), but to date none has been causally related with an understanding of specific molecular links.Familial advanced sleep phase (FASP) is a human behavioral phenotype defined by early sleep time and early morning awakening (15). We previously identified mutations in core clock genes that cause FASP by linkage analysis/positional cloning (16) and candidate gene sequencing (17, 18). Here we identify two rare missense variants in PER3 (PER3-P415A/H417R) that cause FASP and are associated with elevated Beck Depression Inventory (BDI) and seasonality scores. Transgenic mice carrying human PER3-P415A/H417R exhibit delayed phase in a short photoperiod and a lengthened period of wheel-running rhythms in constant light. At a molecular level, the rare variants lead to decreased PER3 protein levels, likely due to decreased protein stability. Moreover, we found that PER3-P415A/H417R can exert effects on the clock (at least in part) by reducing its stabilizing effect on PER1 and PER2. Although hPER3-P415A/H417R transgenic mice display mild measures of depression-like phenotype, Per3^−/− mice exhibit consistent depression-like behaviors in multiple tests. The differences are particularly evident in short photoperiods, implying a role for PER3 in mood regulation. Taken together, these results support a role for PER3 in modulating circadian clock and mood that may be especially critical under conditions of short photoperiod (e.g., during the winter season).     

Neuropsin (OPN5)-mediated photoentrainment of local circadian oscillators in mammalian retina and cornea

The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength–sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.Most mammalian tissues contain autonomous circadian clocks that are synchronized by the suprachiasmatic nuclei (SCN) in the brain (1). The SCN clock itself is entrained by external light cycles through retinal rods, cones, and melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) (2, 3). The retina also manifests a local circadian clock, which regulates many important functions, such as photoreceptor disk shedding, photoreceptor gap-junction coupling, and neurotransmitter release (4–6). Surprisingly, local retinal photoentrainment does not require the SCN, and it also does not require rods, cones, or ipRGCs (7, 8). Thus, the rd1/rd1;Opn4^−/− mouse retina, which has lost essentially all rods and cones due to degeneration and also has an ablated Opn4 gene (3), remains synchronized to light/dark cycles both in vivo and ex vivo (7).To determine the photopigment(s) responsible for local circadian entrainment in the retina, we took a candidate gene approach. Because some cone nuclei may persist in degenerate rd1/rd1 retinas (9), and murine short-wavelength–sensitive cone opsin (OPN1SW) has been reported to be present in the ganglion cell layer (10), we tested the necessity of this pigment for local retinal circadian photoentrainment. We also tested for the involvement of two orphan pigments, encephalopsin (OPN3) (11) and neuropsin (OPN5) (12), both of which are expressed in mammalian retina and, when expressed heterologously, form light-sensitive pigments that activate G proteins (13–17). The function of OPN3 in mammals is unknown despite its widespread expression in neural tissues (18). OPN5 appears to be a deep-brain photopigment in the hypothalamus of birds and is thought to contribute to seasonal reproduction (19–22); it has been immunolocalized to the mammalian inner retina (13, 16) (SI Text); however, to date, no retinal function for this mammalian pigment has been identified. We did not examine two other pigments, retinal G protein-coupled receptor (RGR) opsin (23) and peropsin (RRH) (24). RGR opsin participates in retinoid turnover (25, 26), whereas RRH is expressed principally in the retinal pigment epithelium (24), a cell layer absent in the photoentrainable ex vivo retina preparation (7).