Ubiquinone (UQ), the only known electron carrier in the mammalian electron transport chain (ETC), preferentially delivers electrons to the terminal electron acceptor oxygen (O2). In hypoxia, ubiquinol (UQH2) diverts these electrons onto fumarate instead. Here, we identify rhodoquinone (RQ), an electron carrier detected in mitochondria purified from certain mouse and human tissues that preferentially delivers electrons to fumarate through the reversal of succinate dehydrogenase, independent of environmental O2 levels. The RQ/fumarate ETC is strictly present in vivo and is undetectable in cultured mammalian cells. Using genetic and pharmacologic tools that reprogram the ETC from the UQ/O2 to the RQ/fumarate pathway, we establish that these distinct ETCs support unique programs of mitochondrial function and that RQ confers protection upon hypoxia exposure in vitro and in vivo. Thus, in discovering the presence of RQ in mammals, we unveil a tractable therapeutic strategy that exploits flexibility in the ETC to ameliorate hypoxia-related conditions.

The genetic information stored in mRNAs is decoded by ribosomes during mRNA translation. mRNAs are typically translated by multiple ribosomes simultaneously, but it is unclear whether and how the activity of different ribosomes on an mRNA is coordinated. Here, we develop an imaging approach based on stopless-ORF circular RNAs (socRNAs) to monitor translation of individual ribosomes in either monosomes or polysomes with very high resolution. Using experiments and simulations, we find that translating ribosomes frequently undergo transient collisions. However, unlike persistent collisions, such transient collisions escape detection by cellular quality control pathways. Rather, transient ribosome collisions promote productive translation by reducing ribosome pausing on problematic sequences, a process we term ribosome cooperativity. Ribosome cooperativity also reduces recycling of ribosomes by quality control pathways, thus enhancing processive translation. Together, our single-ribosome imaging approach reveals that ribosomes cooperate during translation to ensure fast and efficient translation.

Coronavirus fusion with and entry into the host cell depends on viral spike, which acts as a crucial component of viral infection. However, the lack of receptor-activated spike intermediate conformation has hindered a comprehensive understanding of spike-induced membrane fusion. Here, we captured an angiotensin-converting enzyme 2 (ACE2)-induced early fusion intermediate conformation (E-FIC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in which heptad repeat 1 (HR1) in S2 has ejected while S1 remains attached. This E-FIC can transition to the late FIC after S2' cleavage. Leveraging this discovery, we designed an E-FIC-targeted dual-functional antiviral protein, AL5E. AL5E effectively inactivated ACE2-using coronaviruses and inhibited their infection, outperforming a mono-functional antiviral in protecting animals against these coronaviruses. This study has identified the E-FIC and used it as a target for the development of a dual-functional antiviral for the prevention and treatment of ACE2-using coronavirus infection.

Human accelerated regions (HARs) have been implicated in human brain evolution. However, insight into the genes and pathways they control is lacking, hindering the understanding of their function. Here, we identify 2,963 conserved gene targets for 1,590 HARs and their orthologs in human and chimpanzee neural stem cells (NSCs). Conserved gene targets are enriched for neurodevelopmental functions and are overrepresented among differentially expressed genes (DEGs) identified in human NSCs (hNSCs) and chimpanzee NSCs (cNSCs) as well as in human versus non-human primate brains. Species-specific gene targets do not converge on any function and are not enriched among DEGs. HAR targets also show cell-type-specific expression in the human fetal brain, including in outer radial glia, which are linked to cortical expansion. Our findings support that HARs influence brain evolution by altering the expression of ancestral gene targets shared between human and chimpanzee rather than by gaining new targets in human and facilitate hypothesis-directed studies of HAR biology.

Androgens, such as 5α-dihydrotestosterone (5α-DHT), regulate numerous functions by binding to nuclear androgen receptors (ARs) and potential unknown membrane receptors. Here, we report that the androgen 5α-DHT activates membrane receptor GPR133 in muscle cells, thereby increasing intracellular cyclic AMP (cAMP) levels and enhancing muscle strength. Further cryoelectron microscopy (cryo-EM) structural analysis of GPR133-Gs in complex with 5α-DHT or its derivative methenolone (MET) reveals the structural basis for androgen recognition. Notably, the presence of the "Φ(F/L)2.64-F3.40-W6.53" and the "F7.42××N/D7.46" motifs, which recognize the hydrophobic steroid core and polar groups, respectively, are common in adhesion GPCRs (aGPCRs), suggesting that many aGPCRs may recognize different steroid hormones. Finally, we exploited in silico screening methods to identify a small molecule, AP503, which activates GPR133 and separates the beneficial muscle-strengthening effects from side effects mediated by AR. Thus, GPR133 represents an androgen membrane receptor that contributes to normal androgen physiology and has important therapeutic potentials.

Knowledge of protein-metabolite interactions can enhance mechanistic understanding and chemical probing of biochemical processes, but the discovery of endogenous ligands remains challenging. Here, we combined rapid affinity purification with precision mass spectrometry and high-resolution molecular docking to precisely map the physical associations of 296 chemically diverse small-molecule metabolite ligands with 69 distinct essential enzymes and 45 transcription factors in the gram-negative bacterium Escherichia coli. We then conducted systematic metabolic pathway integration, pan-microbial evolutionary projections, and independent in-depth biophysical characterization experiments to define the functional significance of ligand interfaces. This effort revealed principles governing functional crosstalk on a network level, divergent patterns of binding pocket conservation, and scaffolds for designing selective chemical probes. This structurally resolved ligand interactome mapping pipeline can be scaled to illuminate the native small-molecule networks of complete cells and potentially entire multi-cellular communities.

Nanoparticle vaccines displaying combinations of SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) could protect against SARS-CoV-2 variants and spillover of zoonotic sarbecoviruses into humans. Using a computational approach, we designed variants of SARS-CoV-2 RBDs and selected 7 natural sarbecovirus RBDs, each predicted to fold properly and abrogate antibody responses to variable epitopes. RBDs were attached to 60-mer nanoparticles to make immunogens displaying two (mosaic-2COMs), five (mosaic-5COM), or seven (mosaic-7COM) different RBDs for comparisons with mosaic-8b, which elicited cross-reactive antibodies and protected animals from sarbecovirus challenges. Naive and COVID-19 pre-vaccinated mice immunized with mosaic-7COM elicited antibodies targeting conserved RBD epitopes, and their sera exhibited higher binding and neutralization titers against sarbecoviruses than mosaic-8b. Mosaic-2COMs and mosaic-5COM elicited higher antibody potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons, supporting its use to protect against SARS-CoV-2 variants and zoonotic sarbecoviruses.

Most land plants form symbioses with microbes to acquire nutrients but also must restrict infection by pathogens. Here, we show that a single pair of lysin-motif-containing receptor-like kinases, MpaLYR and MpaCERK1, mediates both immunity and symbiosis in the liverwort Marchantia paleacea. MpaLYR has a higher affinity for long-chain (CO7) versus short-chain chitin oligomers (CO4). Although both CO7 and CO4 can activate symbiosis-related genes, CO7 triggers stronger immune responses than CO4 in a dosage-dependent manner. CO4 can inhibit CO7-induced strong immune responses, recapitulating the early response to inoculation with the symbiont arbuscular mycorrhizal fungi. We show that phosphate starvation of plants increases their production of strigolactone, which stimulates CO4/CO5 secretion from mycorrhizal fungi, thereby prioritizing symbiosis over immunity. Thus, a single pair of LysM receptors mediates dosage-dependent perception of different chitin oligomers to discern symbiotic and pathogenic microbes in M. paleacea, which may facilitate terrestrialization.

Understanding mammalian preimplantation development, particularly in humans, at the proteomic level remains limited. Here, we applied our comprehensive solution of ultrasensitive proteomic technology to measure the proteomic profiles of oocytes and early embryos and identified nearly 8,000 proteins in humans and over 6,300 proteins in mice. We observed distinct proteomic dynamics before and around zygotic genome activation (ZGA) between the two species. Integrative analysis with translatomic data revealed extensive divergence between translation activation and protein accumulation. Multi-omic analysis indicated that ZGA transcripts often contribute to protein accumulation in blastocysts. Using mouse embryos, we identified several transcriptional regulators critical for early development, thereby linking ZGA to the first lineage specification. Furthermore, single-embryo proteomics of poor-quality embryos from over 100 patient couples provided insights into preimplantation development failure. Our study may contribute to reshaping the framework of mammalian preimplantation development and opening avenues for addressing human infertility.

The cerebral cortex and hippocampus are crucial brain regions for learning and memory, which depend on activity-induced synaptic plasticity involving N-methyl-ᴅ-aspartate receptors (NMDARs). However, subunit assembly and molecular architecture of endogenous NMDARs (eNMDARs) in the brain remain elusive. Using conformation- and subunit-dependent antibodies, we purified eNMDARs from adult rat cerebral cortex and hippocampus. Three major subtypes of GluN1-N2A-N2B, GluN1-N2B, and GluN1-N2A eNMDARs were resolved by cryoelectron microscopy (cryo-EM) at the resolution up to 4.2 Å. The particle ratio of these three subtypes was 9:7:4, indicating that about half of GluN2A and GluN2B subunits are incorporated into the tri-heterotetramers. Structural analysis revealed the asymmetric architecture of the GluN1-N2A-N2B receptor throughout the extracellular to the transmembrane layers. Moreover, the conformational variations between GluN1-N2B and GluN1-N2A-N2B receptors revealed the distinct biophysical properties across different eNMDAR subtypes. Our findings imply the structural and functional complexity of eNMDARs and shed light on structure-based therapeutic design targeting these eNMDARs in vivo.

Industrialization adversely affects the gut microbiome and predisposes individuals to chronic non-communicable diseases. We tested a microbiome restoration strategy comprising a diet that recapitulated key characteristics of non-industrialized dietary patterns (restore diet) and a bacterium rarely found in industrialized microbiomes (Limosilactobacillus reuteri) in a randomized controlled feeding trial in healthy Canadian adults. The restore diet, despite reducing gut microbiome diversity, enhanced the persistence of L. reuteri strain from rural Papua New Guinea (PB-W1) and redressed several microbiome features altered by industrialization. The diet also beneficially altered microbiota-derived plasma metabolites implicated in the etiology of chronic non-communicable diseases. Considerable cardiometabolic benefits were observed independently of L. reuteri administration, several of which could be accurately predicted by baseline and diet-responsive microbiome features. The findings suggest that a dietary intervention targeted toward restoring the gut microbiome can improve host-microbiome interactions that likely underpin chronic pathologies, which can guide dietary recommendations and the development of therapeutic and nutritional strategies.

Biocatalytic cascades with spatial proximity can orchestrate multistep pathways to form metabolic highways, which enhance the overall catalytic efficiency. However, the effect of spatial organization on catalytic activity is poorly understood, and multienzyme architectural engineering with predictable performance remains unrealized. Here, we developed a standardized framework, called iMARS, to rapidly design the optimal multienzyme architecture by integrating high-throughput activity tests and structural analysis. The approach showed potential for industrial-scale applications, with artificial fusion enzymes designed by iMARS significantly improving the production of resveratrol by 45.1-fold and raspberry ketone by 11.3-fold in vivo, as well as enhancing ergothioneine synthesis in fed-batch fermentation. In addition, iMARS greatly enhanced the in vitro catalytic efficiency of the multienzyme complexes for PET plastic depolymerization and vanillin biosynthesis. As a generalizable and flexible strategy at molecular level, iMARS could greatly facilitate green chemistry, synthetic biology, and biomanufacturing.

The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20- to 25-nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We find that CENP-C is required in mitosis, not just for kinetochore assembly, likely reflecting its role in organizing the inner kinetochore during chromosome segregation. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils extending >1 μm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.

Despite recent advances in imaging- and antibody-based methods, achieving in-depth, high-resolution protein mapping across entire tissues remains a significant challenge in spatial proteomics. Here, we present parallel-flow projection and transfer learning across omics data (PLATO), an integrated framework combining microfluidics with deep learning to enable high-resolution mapping of thousands of proteins in whole tissue sections. We validated the PLATO framework by profiling the spatial proteome of the mouse cerebellum, identifying 2,564 protein groups in a single run. We then applied PLATO to rat villus and human breast cancer samples, achieving a spatial resolution of 25 μm and uncovering proteomic dynamics associated with disease states. This approach revealed spatially distinct tumor subtypes, identified key dysregulated proteins, and provided novel insights into the complexity of the tumor microenvironment. We believe that PLATO represents a transformative platform for exploring spatial proteomic regulation and its interplay with genetic and environmental factors.

Viruses encode proteins that inhibit host defenses, but sifting through the millions of available viral sequences for immune-modulatory proteins has been so far impractical. Here, we develop a process to systematically screen virus-encoded proteins for inhibitors that physically bind host immune proteins. Focusing on Thoeris and CBASS, bacterial defense systems that are the ancestors of eukaryotic Toll/interleukin-1 receptor (TIR) and cyclic GMP-AMP synthase (cGAS) immunity, we discover seven families of Thoeris and CBASS inhibitors, encompassing thousands of genes widespread in phages. Verified inhibitors exhibit extensive physical interactions with the respective immune protein counterpart, with all inhibitors blocking the active site of the immune protein. Remarkably, a phage-encoded inhibitor of bacterial TIR proteins can bind and inhibit distantly related human and plant immune TIRs, and a phage-derived inhibitor of bacterial cGAS-like enzymes can inhibit the human cGAS. Our results demonstrate that phages are a reservoir for immune-modulatory proteins capable of inhibiting bacterial, animal, and plant immunity.

Plasma membrane rupture during lytic cell death was previously believed to occur through passive osmosis that burst open the membrane. Recent publications, including one in this issue of Cell, suggest that plasma membrane rupture is an active process mediated by ninjurin-1 (NINJ1) oligomers that dissolve membranes and/or assemble large pores.

The factors shaping human microbiome variation are a major focus of biomedical research. While other fields have used large sequencing compendia to extract insights requiring otherwise impractical sample sizes, the microbiome field has lacked a comparably sized resource for the 16S rRNA gene amplicon sequencing commonly used to quantify microbiome composition. To address this gap, we processed 168,464 publicly available human gut microbiome samples with a uniform pipeline. We use this compendium to evaluate geographic and technical effects on microbiome variation. We find that regions such as Central and Southern Asia differ significantly from the more thoroughly characterized microbiomes of Europe and Northern America and that composition alone can be used to predict a sample's region of origin. We also find strong associations between microbiome variation and technical factors such as primers and DNA extraction. We anticipate this growing work, the Human Microbiome Compendium, will enable advanced applied and methodological research.