Gpr35 shapes gut microbial ecology to modulate hepatic steatosis.

The gut microbiome is closely shaped by host genetic and dietary factors to regulate metabolic health and disease. However, the signaling mechanisms underlying such interactions have been largely unclear. Here we identify G protein-coupled receptor 35 (Gpr35) as a regulator of gut microbial ecology and the susceptibility to obesity and hepatic steatosis in mice. Both global and intestinal epithelia specific ablation of Gpr35 aggravated high-fat diet (HFD)-induced metabolic disturbance and hepatic steatosis in mice. Gpr35 deficiency induced a remarkable loss of goblet cells and an extensive remodeling of the gut microbiome, featuring enrichment of the Bacteroides and Ruminococcus genera. Antibiotics treatment and co-housing alleviated the metabolic disturbance markers in Gpr35 deficient mice. Spatiotemporal profiling and mono-colonization screening revealed that Ruminococcus gnavus synergized with HFD to promote hepatic steatosis possibly via tryptophan and phenylalanine pathway metabolites. Our results provide mechanistic insights into a genetic-diet-microbe interplay that dictates susceptibility to metabolic disorder.

Anti-diabetic drugs and sarcopenia: emerging links, mechanistic insights, and clinical implications.

Sarcopenia, characterized by loss of skeletal muscle mass, quality, and strength, has become a common hallmark of ageing and many chronic diseases. Diabetes mellitus patients have a higher prevalence of sarcopenia, which greatly aggravates the metabolic disturbance and compromises treatment response. Preclinical and clinical studies have shown differential impacts of anti-diabetic drugs on skeletal muscle mass, strength, and performance, highlighting the importance of rational therapeutic regimen from the perspective of sarcopenia risk. In this review, we provide an update on the regulation of muscle mass and quality by major anti-diabetic drugs, focusing primarily on emerging data from clinical studies. We also discuss the underlying mechanisms and clinical implications for optimal selection of anti-diabetic drugs to reduce the risk of sarcopenia. In view of the lifelong use of anti-diabetic drugs, we propose that a better understanding of the sarcopenia risk and interventional strategies is worthy of attention in future studies.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins.

Massively parallel unsupervised single-particle cryo-EM data clustering via statistical manifold learning.

Structural heterogeneity in single-particle cryo-electron microscopy (cryo-EM) data represents a major challenge for high-resolution structure determination. Unsupervised classification may serve as the first step in the assessment of structural heterogeneity. However, traditional algorithms for unsupervised classification, such as K-means clustering and maximum likelihood optimization, may classify images into wrong classes with decreasing signal-to-noise-ratio (SNR) in the image data, yet demand increased computational costs. Overcoming these limitations requires further development of clustering algorithms for high-performance cryo-EM data processing. Here we introduce an unsupervised single-particle clustering algorithm derived from a statistical manifold learning framework called generative topographic mapping (GTM). We show that unsupervised GTM clustering improves classification accuracy by about 40% in the absence of input references for data with lower SNRs. Applications to several experimental datasets suggest that our algorithm can detect subtle structural differences among classes via a hierarchical clustering strategy. After code optimization over a high-performance computing (HPC) environment, our software implementation was able to generate thousands of reference-free class averages within hours in a massively parallel fashion, which allows a significant improvement on ab initio 3D reconstruction and assists in the computational purification of homogeneous datasets for high-resolution visualization.

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5 Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly.

Structural basis for dynamic regulation of the human 26S proteasome.

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near-atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism.

Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of the human caspase-1 CARD domain (caspase-1(CARD)) filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins: human inhibitor of CARD (INCA or CARD17) and ICEBERG (CARD18). Our results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on caspase-1(CARD) polymerization in vitro and inflammasome activation in cells. Whereas caspase-1(CARD) uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces and thus terminates the caspase-1 filament.

Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly.

Mathematical modeling of genetic circuits generally assumes that gene expression is at steady state when measurements are performed. However, conventional methods of measurement do not necessarily guarantee that this assumption is satisfied. In this study, we reveal a bi-plateau mode of gene expression at the single-cell level in bacterial batch cultures. The first plateau is dynamically active, where gene expression is at steady state; the second plateau, however, is dynamically inactive. We further demonstrate that the predictability of assembled genetic circuits in the first plateau (steady state) is much higher than that in the second plateau where conventional measurements are often performed. By taking the nature of steady state into consideration, our method of measurement promises to directly capture the intrinsic property of biological parts/circuits regardless of circuit-host or circuit-environment interactions.

Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization.

The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo-electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes.

Design, construction, and characterization of a set of biosensors for aromatic compounds.

Aromatic pollutants in the environments pose significant threat to human health due to their persistence and toxicity. Here, we report the design and comprehensive characterization of a set of aromatic biosensors constructed using green fluorescence protein as the reporter and aromatics-responsive transcriptional regulators, namely, NahR, XylS, HbpR, and DmpR, as the detectors. The genetic connections between the detectors and the reporter were carefully adjusted to achieve fold inductions far exceeding those reported in previous studies. For each biosensor, the functional characteristics including the dose-responses, dynamic range, and the detection spectrum of aromatic species were thoroughly measured. In particular, the interferences that nontypical inducers exert on each biosensor's response to its strongest inducer were evaluated. These well-characterized biosensors might serve as potent tools for environmental monitoring as well as quantitative gene regulation.

Automated design of genetic toggle switches with predetermined bistability.

Synthetic biology aims to rationally construct biological devices with required functionalities. Methods that automate the design of genetic devices without post-hoc adjustment are therefore highly desired. Here we provide a method to predictably design genetic toggle switches with predetermined bistability. To accomplish this task, a biophysical model that links ribosome binding site (RBS) DNA sequence to toggle switch bistability was first developed by integrating a stochastic model with RBS design method. Then, to parametrize the model, a library of genetic toggle switch mutants was experimentally built, followed by establishing the equivalence between RBS DNA sequences and switch bistability. To test this equivalence, RBS nucleotide sequences for different specified bistabilities were in silico designed and experimentally verified. Results show that the deciphered equivalence is highly predictive for the toggle switch design with predetermined bistability. This method can be generalized to quantitative design of other probabilistic genetic devices in synthetic biology.