BCAA-nitrogen flux in brown fat controls metabolic health independent of thermogenesis.

Brown adipose tissue (BAT) is best known for thermogenesis. Rodent studies demonstrated that enhanced BAT thermogenesis is tightly associated with increased energy expenditure, reduced body weight, and improved glucose homeostasis. However, human BAT is protective against type 2 diabetes, independent of body weight. The mechanism underlying this dissociation remains unclear. Here, we report that impaired mitochondrial catabolism of branched-chain amino acids (BCAAs) in BAT, by deleting mitochondrial BCAA carriers (MBCs), caused systemic insulin resistance without affecting energy expenditure and body weight. Brown adipocytes catabolized BCAA in the mitochondria as nitrogen donors for the biosynthesis of non-essential amino acids and glutathione. Impaired mitochondrial BCAA-nitrogen flux in BAT resulted in increased oxidative stress, decreased hepatic insulin signaling, and decreased circulating BCAA-derived metabolites. A high-fat diet attenuated BCAA-nitrogen flux and metabolite synthesis in BAT, whereas cold-activated BAT enhanced the synthesis. This work uncovers a metabolite-mediated pathway through which BAT controls metabolic health beyond thermogenesis.

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Membraneless channels sieve cations in ammonia-oxidizing marine archaea.

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.

Capturing carbon dioxide from air with charged-sorbents.

Emissions reduction and greenhouse gas removal from the atmosphere are both necessary to achieve net-zero emissions and limit climate change1. There is thus a need for improved sorbents for the capture of carbon dioxide from the atmosphere, a process known as direct air capture. In particular, low-cost materials that can be regenerated at low temperatures would overcome the limitations of current technologies. In this work, we introduce a new class of designer sorbent materials known as 'charged-sorbents'. These materials are prepared through a battery-like charging process that accumulates ions in the pores of low-cost activated carbons, with the inserted ions then serving as sites for carbon dioxide adsorption. We use our charging process to accumulate reactive hydroxide ions in the pores of a carbon electrode, and find that the resulting sorbent material can rapidly capture carbon dioxide from ambient air by means of (bi)carbonate formation. Unlike traditional bulk carbonates, charged-sorbent regeneration can be achieved at low temperatures (90-100 °C) and the sorbent's conductive nature permits direct Joule heating regeneration2,3 using renewable electricity. Given their highly tailorable pore environments and low cost, we anticipate that charged-sorbents will find numerous potential applications in chemical separations, catalysis and beyond.

Identification of multipotent luminal progenitor cells in human prostate organoid cultures.

The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.

Organoid cultures derived from patients with advanced prostate cancer.

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Whole-exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection.

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA.

Structure, substrate recognition and initiation of hyaluronan synthase.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.

Author Correction: Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

Activator-induced conformational changes regulate division-associated peptidoglycan amidases.

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

The genetic architecture of pediatric cardiomyopathy.

To understand the genetic contribution to primary pediatric cardiomyopathy, we performed exome sequencing in a large cohort of 528 children with cardiomyopathy. Using clinical interpretation guidelines and targeting genes implicated in cardiomyopathy, we identified a genetic cause in 32% of affected individuals. Cardiomyopathy sub-phenotypes differed by ancestry, age at diagnosis, and family history. Infants < 1 year were less likely to have a molecular diagnosis (p < 0.001). Using a discovery set of 1,703 candidate genes and informatic tools, we identified rare and damaging variants in 56% of affected individuals. We see an excess burden of damaging variants in affected individuals as compared to two independent control sets, 1000 Genomes Project (p < 0.001) and SPARK parental controls (p < 1 × 10-16). Cardiomyopathy variant burden remained enriched when stratified by ancestry, variant type, and sub-phenotype, emphasizing the importance of understanding the contribution of these factors to genetic architecture. Enrichment in this discovery candidate gene set suggests multigenic mechanisms underlie sub-phenotype-specific causes and presentations of cardiomyopathy. These results identify important information about the genetic architecture of pediatric cardiomyopathy and support recommendations for clinical genetic testing in children while illustrating differences in genetic architecture by age, ancestry, and sub-phenotype and providing rationale for larger studies to investigate multigenic contributions.

U-Pb-dated flowstones restrict South African early hominin record to dry climate phases.

The Cradle of Humankind (Cradle) in South Africa preserves a rich collection of fossil hominins representing Australopithecus, Paranthropus and Homo1. The ages of these fossils are contentious2-4 and have compromised the degree to which the South African hominin record can be used to test hypotheses of human evolution. However, uranium-lead (U-Pb) analyses of horizontally bedded layers of calcium carbonate (flowstone) provide a potential opportunity to obtain a robust chronology5. Flowstones are ubiquitous cave features and provide a palaeoclimatic context, because they grow only during phases of increased effective precipitation6,7, ideally in closed caves. Here we show that flowstones from eight Cradle caves date to six narrow time intervals between 3.2 and 1.3 million years ago. We use a kernel density estimate to combine 29 U-Pb ages into a single record of flowstone growth intervals. We interpret these as major wet phases, when an increased water supply, more extensive vegetation cover and at least partially closed caves allowed for undisturbed, semi-continuous growth of the flowstones. The intervening times represent substantially drier phases, during which fossils of hominins and other fossils accumulated in open caves. Fossil preservation, restricted to drier intervals, thus biases the view of hominin evolutionary history and behaviour, and places the hominins in a community of comparatively dry-adapted fauna. Although the periods of cave closure leave temporal gaps in the South African fossil record, the flowstones themselves provide valuable insights into both local and pan-African climate variability.

BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Lung Injury Prediction Model in Bone Marrow Transplantation: A Multicenter Cohort Study.

Rationale: Pulmonary complications contribute significantly to nonrelapse mortality following hematopoietic stem cell transplantation (HCT). Identifying patients at high risk can help enroll such patients into clinical studies to better understand, prevent, and treat posttransplantation respiratory failure syndromes. Objectives: To develop and validate a prediction model to identify those at increased risk of acute respiratory failure after HCT. Methods: Patients underwent HCT between January 1, 2019, and December 31, 2021, at one of three institutions. Those treated in Rochester, MN, formed the derivation cohort, and those treated in Scottsdale, AZ, or Jacksonville, FL, formed the validation cohort. The primary outcome was the development of acute respiratory distress syndrome (ARDS), with secondary outcomes including the need for invasive mechanical ventilation (IMV) and/or noninvasive ventilation (NIV). Predictors were based on prior case-control studies. Measurements and Main Results: Of 2,450 patients undergoing stem cell transplantation, there were 1,718 hospitalizations (888 patients) in the training cohort and 1,005 hospitalizations (470 patients) in the test cohort. A 22-point model was developed, with 11 points from prehospital predictors and 11 points from posttransplantation or early (<24-h) in-hospital predictors. The model performed well in predicting ARDS (C-statistic, 0.905; 95% confidence interval [CI], 0.870-0.941) and the need for IMV and/or NIV (C-statistic, 0.863; 95% CI, 0.828-0.898). The test cohort differed markedly in demographic, medical, and hematologic characteristics. The model also performed well in this setting in predicting ARDS (C-statistic, 0.841; 95% CI, 0.782-0.900) and the need for IMV and/or NIV (C-statistic, 0.872; 95% CI, 0.831-0.914). Conclusions: A novel prediction model incorporating data elements from the pretransplantation, posttransplantation, and early in-hospital domains can reliably predict the development of post-HCT acute respiratory failure.