Protein restriction and branched‐chain amino acid restriction promote geroprotective shifts in metabolism

The proportion of humans suffering from age‐related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie—and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched‐chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality—the specific composition of dietary protein—may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.     

Male castration increases adiposity via small intestinal microbial alterations

Castration of young males is widely used in the cattle industry to improve meat quality, but the mechanism linking hypogonadism and host metabolism is not clear. Here, we use metataxonomic and metabolomic approaches to evaluate the intestinal microbiota and host metabolism in male, castrated male (CtM), and female cattle. After pubescence, the CtM cattle harbor distinct ileal microbiota dominated by the family Peptostreptococcaceae and exhibit distinct serum and muscle amino acid profiles (i.e., highly abundant branched‐chain amino acids), with increased extra‐ and intramuscular fat storage. We also evaluate the causative factor(s) that underpin the alteration of the intestinal microbiota and host metabolic phenotype in response to hypogonadism. Castration of male mice phenocopies both the intestinal microbial alterations and obese‐prone metabolism observed in cattle. Antibiotic treatment and fecal microbiota transplantation experiments in a mouse model confirm that the intestinal microbial alterations associated with hypogonadism are a key contributor to the obese phenotype in the CtM animals. Collectively, targeting the gut microbiota is a potential therapeutic strategy for the treatment of both hypogonadism and obesity.     

A heterotypic assembly mechanism regulates CHIP E3 ligase activity

CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation.     

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.     

MIG‐6 is essential for promoting glucose metabolic reprogramming and tumor growth in triple‐negative breast cancer

Treatment of triple‐negative breast cancer (TNBC) remains challenging due to a lack of effective targeted therapies. Dysregulated glucose uptake and metabolism are essential for TNBC growth. Identifying the molecular drivers and mechanisms underlying the metabolic vulnerability of TNBC is key to exploiting dysregulated cancer metabolism for therapeutic applications. Mitogen‐inducible gene‐6 (MIG‐6) has long been thought of as a feedback inhibitor that targets activated EGFR and suppresses the growth of tumors driven by constitutive activated mutant EGFR. Here, our bioinformatics and histological analyses uncover that MIG‐6 is upregulated in TNBC and that MIG‐6 upregulation is positively correlated with poorer clinical outcomes in TNBC. Metabolic arrays and functional assays reveal that MIG‐6 drives glucose metabolism reprogramming toward glycolysis. Mechanistically, MIG‐6 recruits HAUSP deubiquitinase for stabilizing HIF1α protein expression and the subsequent upregulation of GLUT1 and other HIF1α‐regulated glycolytic genes, substantiating the comprehensive regulation of MIG‐6 in glucose metabolism. Moreover, our mouse studies demonstrate that MIG‐6 regulates GLUT1 expression in tumors and subsequent tumor growth in vivo. Collectively, this work reveals that MIG‐6 is a novel prognosis biomarker, metabolism regulator, and molecular driver of TNBC.     

N‐acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS‐low, glutaminolysis‐high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N‐acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL‐10, enforces GS expression in macrophages. In turn, GS‐high macrophages acquire M2‐like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2‐like macrophage phenotype, IL‐10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti‐inflammatory, protumoral function of NAA.     

Genome‐scale metabolic modeling reveals SARS‐CoV‐2‐induced metabolic changes and antiviral targets

Tremendous progress has been made to control the COVID‐19 pandemic caused by the SARS‐CoV‐2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS‐CoV‐2 infection using genome‐scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS‐CoV‐2 infection. We next applied the GEM‐based metabolic transformation algorithm to predict anti‐SARS‐CoV‐2 targets that counteract the virus‐induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco‐2 cells. Further generating and analyzing RNA‐sequencing data of remdesivir‐treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti‐SARS‐CoV‐2 drug. Our study provides clinical data‐supported candidate anti‐SARS‐CoV‐2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.     

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1

Knowledge of a protein’s spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3‐dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long‐lasting immunosuppression. Whether the two activities—namely, the catalytic and signaling functions—are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3‐kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1‐dependent signaling events. Thus, IDO1’s spatial dynamics meet the needs for short‐acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1‐centered therapy in inflammation and autoimmunity.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

Cyclic di‐GMP modulates sessile‐motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice‐growing countries. We showed recently that the universal bacterial second messenger c‐di‐GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c‐di‐GMP receptor domain, known as the PilZ‐domain. By deleting all the genes encoding c‐di‐GMP‐degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c‐di‐GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild‐type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c‐di‐GMP, which together play a critical role in regulating the c‐di‐GMP‐associated functionality. The findings from this study fill a gap in our understanding of how c‐di‐GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.     

The genotype‐phenotype landscape of an allosteric protein

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype‐phenotype relationships remains elusive. Here, we report the large‐scale measurement of the genotype‐phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long‐read and short‐read DNA sequencing, we quantitatively measure the dose‐response curves for nearly 10⁵ variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence‐structure‐function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural‐network models, but unpredictable changes also occur. For example, we were surprised to discover a new band‐stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.     

Incomplete proline catabolism drives premature sperm aging

Infertility is an increasingly common health issue, with rising prevalence in advanced parental age. Environmental stress has established negative effects on reproductive health, however, the impact of altering cellular metabolism and its endogenous reactive oxygen species (ROS) on fertility remains unclear. Here, we demonstrate the loss of proline dehydrogenase, the first committed step in proline catabolism, is relatively benign. In contrast, disruption of alh‐6, which facilitates the second step of proline catabolism by converting 1‐pyrroline‐5‐carboxylate (P5C) to glutamate, results in premature reproductive senescence, specifically in males. The premature reproductive senescence in alh‐6 mutant males is caused by aberrant ROS homeostasis, which can be countered by genetically limiting the first committed step of proline catabolism that functions upstream of ALH‐6 or by pharmacological treatment with antioxidants. Taken together, our work uncovers proline metabolism as a critical component of normal sperm function that can alter the rate of aging in the male reproductive system.     

Evolution of homo‐oligomerization of methionine S‐adenosyltransferases is replete with structure–function constrains

Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S‐adenosyltransferases (MATs)—dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer‐dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate‐induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.     

Gel‐like inclusions of C‐terminal fragments of TDP‐43 sequester stalled proteasomes in neurons

Aggregation of the multifunctional RNA‐binding protein TDP‐43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C‐terminal fragments of ~25 kDa ("TDP‐25") accumulate in cytoplasmic inclusions. Here, we analyze gain‐of‐function mechanisms of TDP‐25 combining cryo‐electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP‐25 inclusions are amorphous, and photobleaching experiments reveal gel‐like biophysical properties that are less dynamic than nuclear TDP‐43. Compared with full‐length TDP‐43, the TDP‐25 interactome is depleted of low‐complexity domain proteins. TDP‐25 inclusions are enriched in 26S proteasomes adopting exclusively substrate‐processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP‐25 impairs proteostasis, and this inhibitory function is enhanced by ALS‐causing TDP‐43 mutations. These findings support a patho‐physiological relevance of proteasome dysfunction in ALS/FTD.     

Thermodynamic coupling between neighboring binding sites in homo‐oligomeric ligand sensing proteins from mass resolved ligand‐dependent population distributions

Homo‐oligomeric ligand‐activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand‐binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring‐shaped homo‐oligomeric trp RNA‐binding attenuation protein (TRAP). First, we developed a nearest‐neighbor statistical thermodynamic binding model comprising microscopic free energies for ligand binding to isolated sites ΔG ₀, and for coupling between adjacent sites, ΔG _α . Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2 ^N possible liganded states. We then experimentally monitored ligand‐dependent population shifts using conventional spectroscopic and calorimetric methods and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand‐dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2 ^N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure–thermodynamic linkage that drives cooperativity.