Search

Summary: Calorie restriction (CR) extends lifespan and healthspan in diverse species. Comparing ad libitum- and CR-fed mice is challenging due to their significantly different feeding patterns, with CR-fed mice consuming their daily meal in 2 h and then subjecting themselves to a prolonged daily fast. Here, we examine how ad libitum- and CR-fed mice respond to tests performed at various times and fasting durations and find that the effects of CR—insulin sensitivity, circulating metabolite levels, and mechanistic target of rapamycin 1 (mTORC1) activity—result from the specific temporal conditions chosen, with CR-induced improvements in insulin sensitivity observed only after a prolonged fast, and the observed differences in mTORC1 activity between ad libitum- and CR-fed mice dependent upon both fasting duration and the specific tissue examined. Our results demonstrate that much of our understanding of the effects of CR are related to when, relative to feeding, we choose to examine the mice.

To molecularly characterize the impact of exercise on mitigating neoadjuvant treatment (NAT)-induced physical decline in pancreatic ductal adenocarcinoma (PDAC) patients, a multi-omics approach was employed for the analysis of plasma samples before and after a personalized exercise intervention. Consisting of personalized aerobic and resistance exercises, this intervention was associated with significant molecular changes that correlated with improvements in lean mass, appendicular skeletal muscle index (ASMI), and performance in the 400-m walk test (MWT) and sit-to-stand test. These alterations indicated exercise-induced modulation of inflammation and mitochondrial function markers. This case study provides proof-of-principal application for multiomics-based assessments of supervised exercise, thereby supporting this intervention as a feasible and beneficial intervention for PDAC patients to potentially enhance treatment response and patient quality of life. The molecular changes observed here underscore the importance of physical activity in cancer treatment protocols, advocating for the development of accessible multiomics-guided exercise programs for cancer patients.

Not available.

Summary: The lymphatic fluid is the conduit by which part of the tissue “omics” is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.

Abstract Aconitate decarboxylase 1 (ACOD1) is the enzyme synthesizing itaconate, an immuno-regulatory metabolite tuning host-pathogen interactions. Such functions are achieved by affecting metabolic pathways regulating inflammation and microbe survival. However, at the whole-body level, metabolic roles of itaconate remain largely unresolved. By using multiomics-integrated approaches, here we show that ACOD1 responds to high-fat diet consumption in mice by promoting gut microbiota alterations supporting metabolic disease. Genetic disruption of itaconate biosynthesis protects mice against obesity, alterations in glucose homeostasis and liver metabolic dysfunctions by decreasing meta-inflammatory responses to dietary lipid overload. Mechanistically, fecal metagenomics and microbiota transplantation experiments demonstrate such effects are dependent on an amelioration of the intestinal ecosystem composition, skewed by high-fat diet feeding towards obesogenic phenotype. In particular, unbiased fecal microbiota profiling and axenic culture experiments point towards a primary role for itaconate in inhibiting growth of Bacteroidaceae and Bacteroides, family and genus of Bacteroidetes phylum, the major gut microbial taxon associated with metabolic health. Specularly to the effects imposed by Acod1 deficiency on fecal microbiota, oral itaconate consumption enhances diet-induced gut dysbiosis and associated obesogenic responses in mice. Unveiling an unrecognized role of itaconate, either endogenously produced or exogenously administered, in supporting microbiota alterations underlying diet-induced obesity in mice, our study points ACOD1 as a target against inflammatory consequences of overnutrition.

Mitapivat, a pyruvate kinase (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Introduction: Human milk delivers critical nutritional and immunological support to human infants. Milk fat globules (MFGs) and their associated membranes (MFGMs) contain the majority of milk lipids and many bioactive components that contribute to neonatal development and health, yet their compositions have not been fully defined, and the mechanisms responsible for formation of these structures remain incompletely understood.Methods: In this study, we used untargeted mass spectrometry to quantitatively profile the protein compositions of freshly obtained MFGs and their paired, physically separated MFGM fractions from 13 human milk samples. We also quantitatively profiled the MFG protein compositions of 9 pooled milk samples from 18 lactating mouse dams.Results: We identified 2,453 proteins and 2,795 proteins in the majority of human MFG and MFGM samples, respectively, and 1,577 proteins in mouse MFGs. Using paired analyses of protein abundance in MFGMs compared to MFGs (MFGM-MFG; 1% FDR), we identified 699 proteins that were more highly abundant in MFGMs (MFGM-enriched), and 201 proteins that were less abundant in MFGMs (cytoplasmic). MFGM-enriched proteins comprised membrane systems (apical plasma membrane and multiple vesicular membranes) hypothesized to be responsible for lipid and protein secretion and components of membrane transport and signaling systems. Cytoplasmic proteins included ribosomal and proteasomal systems. Comparing abundance between human and mouse MFGs, we found a positive correlation (R2 = 0.44, p < 0.0001) in the relative abundances of 1,279 proteins that were found in common across species.Discussion: Comparative pathway enrichment analyses between human and mouse samples reveal similarities in membrane trafficking and signaling pathways involved in milk fat secretion and identify potentially novel immunological components of MFGs. Our results advance knowledge of the composition and relative quantities of proteins in human and mouse MFGs in greater detail, provide a quantitative profile of specifically enriched human MFGM proteins, and identify core cellular systems involved in milk lipid secretion.

Abstract Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor, but the mechanisms underlying tumor growth and progression remain unclear. The protein arginine methyltransferases (PRMTs) regulate a variety of biological processes, however, their roles in GBM growth and progression are not fully understood. In this study, our functional analysis of gene expression networks revealed that among the PRMT family expression of PRMT3 was most significantly enriched in both GBM and low-grade gliomas. Higher PRMT3 expression predicted poorer overall survival rate in patients with gliomas. Knockdown of PRMT3 markedly reduced the proliferation and migration of GBM cell lines and patient-derived glioblastoma stem cells (GSC) in cell culture, while its over-expression increased the proliferative capacity of GSC cells by promoting cell cycle progression. Consistently, stable PRMT3 knockdown strongly inhibited tumor growth in xenograft mouse models, along with a significant decrease in cell proliferation as well as an increase in apoptosis. We further found that PRMT3 reprogrammed metabolic pathways to promote GSC growth via increasing glycolysis and its critical transcriptional regulator HIF1α. In addition, pharmacological inhibition of PRMT3 with a PRMT3-specific inhibitor SGC707 impaired the growth of GBM cells. Thus, our study demonstrates that PRMT3 promotes GBM progression by enhancing HIF1A-mediated glycolysis and metabolic rewiring, presenting a point of metabolic vulnerability for therapeutic targeting in malignant gliomas.

Aberrant coagulation in sickle cell disease (SCD) is linked to extracellular vesicle (EV) exposure. However, there is no consensus on the contributions of small EVs (SEVs) and large EVs (LEVs) toward underlying coagulopathy or on their molecular cargo. The present observational study compared the thrombin potential of SEVs and LEVs isolated from the plasma of stable pediatric and adult SCD patients. Further, EV lipid and protein contents were analyzed to define markers consistent with activation of thrombin and markers of underlying coagulopathy. Results suggested that LEVs—but not SEVs—from pediatrics and adults similarly enhanced phosphatidylserine (PS)-dependent thrombin generation, and cell membrane procoagulant PS (18:0;20:4 and 18:0;18:1) were the most abundant lipids found in LEVs. Further, LEVs showed activated coagulation in protein pathway analyses, while SEVs demonstrated high levels of cholesterol esters and a protein pathway analysis that identified complement factors and inflammation. We suggest that thrombin potential of EVs from both stable pediatric and adult SCD patients is similarly dependent on size and show lipid and protein contents that identify underlying markers of coagulation and inflammation.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

Summary: Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to −3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

Lesch–Nyhan syndrome (LN) is an is an X-linked recessive inborn error of metabolism that arises from a deficiency of purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disease manifests severely, causing intellectual deficits and other neural abnormalities, hypercoagulability, uncontrolled self-injury, and gout. While allopurinol is used to alleviate gout, other symptoms are less understood, impeding treatment. Herein, we present a high-throughput multi-omics analysis of red blood cells (RBCs) from three pediatric siblings carrying a novel S162N HPRT1 mutation. RBCs from both parents—the mother, a heterozygous carrier, and the father, a clinically healthy control—were also analyzed. Global metabolite analysis of LN RBCs shows accumulation of glycolytic intermediates upstream of pyruvate kinase, unsaturated fatty acids, and long chain acylcarnitines. Similarly, highly unsaturated phosphatidylcholines are also elevated in LN RBCs, while free choline is decreased. Intracellular iron, zinc, selenium, and potassium are also decreased in LN RBCs. Global proteomics documented changes in RBC membrane proteins, hemoglobin, redox homeostasis proteins, and the enrichment of coagulation proteins. These changes were accompanied by elevation in protein glutamine deamidation and methylation in the LN children and carrier mother. Treatment with allopurinol incompletely reversed the observed phenotypes in the two older siblings currently on this treatment. This unique data set provides novel opportunities for investigations aimed at potential therapies for LN-associated sequelae.

The broad spectrum of beta-thalassemia (βThal) mutations may result in mild reduction (β++), severe reduction (β+) or complete absence (β0) of beta-globin synthesis. βThal heterozygotes eligible for blood donation are “good storers” in terms of red blood cell (RBC) fragility, proteostasis and redox parameters of storage lesion. However, it has not been examined if heterogeneity in genetic backgrounds among βThal-trait donors affects their RBC storability profile. For this purpose, a paired analysis of physiological and omics parameters was performed in freshly drawn blood and CPD/SAGM-stored RBCs donated by eligible volunteers of β++ (N = 4), β+ (N = 9) and β0 (N = 2) mutation-based phenotypes. Compared to β+, β++ RBCs were characterized by significantly lower RDW and HbA2 but higher hematocrit, MCV and NADPH levels in vivo. Moreover, they had lower levels of reactive oxygen species and markers of oxidative stress, already from baseline. Interestingly, their lower myosin and arginase membrane levels were accompanied by increased cellular fragility and arginine values. Proteostasis markers (proteasomal activity and/or chaperoning-protein membrane-binding) seem to be also diminished in β++ as opposed to the other two phenotypic groups. Overall, despite the low number of samples in the sub-cohorts, it seems that the second level of genetic variability among the group of βThal-trait donors is reflected not only in the physiological features of RBCs in vivo, but almost equally in their storability profiles. Mutations that only slightly affect the globin chain equilibrium direct RBCs towards phenotypes closer to the average control, at least in terms of fragility indices and proteostatic dynamics.

Recent advances in targeting leukemic stem cells (LSCs) using venetoclax with azacitidine (ven + aza) has significantly improved outcomes for de novo acute myeloid leukemia (AML) patients. However, patients who relapse after traditional chemotherapy are often venetoclax-resistant and exhibit poor clinical outcomes. We previously described that fatty acid metabolism drives oxidative phosphorylation (OXPHOS) and acts as a mechanism of LSC survival in relapsed/refractory AML. Here, we report that chemotherapy-relapsed primary AML displays aberrant fatty acid and lipid metabolism, as well as increased fatty acid desaturation through the activity of fatty acid desaturases 1 and 2, and that fatty acid desaturases function as a mechanism of recycling NAD+ to drive relapsed LSC survival. When combined with ven + aza, the genetic and pharmacologic inhibition of fatty acid desaturation results in decreased primary AML viability in relapsed AML. This study includes the largest lipidomic profile of LSC-enriched primary AML patient cells to date and indicates that inhibition of fatty acid desaturation is a promising therapeutic target for relapsed AML.

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS–Hook–FHIP (‘FHF’) cargo adaptor complex links dynein to cargo in humans and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein ‘interactome’ of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 colocalize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A, and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS–Hook–FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

COVID19 is a heterogeneous medical condition involving diverse underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Low antibody titers associate with hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, and depletion of lymphocytes, neutrophils, and platelets. Upon seroconversion, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased D-dimer, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

Red blood cells (RBC) have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which RBC neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how RBC repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to RBC is direct repair of the damaged molecules, as RBC cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. RBC express an L-isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters RBC metabolism in a healthy state, but does not impair the circulatory lifespan of RBC. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, RBC from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

Reticuloendothelial macrophages engulf ∼0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of mouse bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.

Adult stem cells have unique properties in both proliferation and differentiation preference. In this study, we hypothesized that adipose stem cells have a depot-dependent lineage preference. Four rabbits were used to provide donor-matched adipose stem cells from either subcutaneous adipose tissue (ScAT) or infrapatellar fat pad (IPFP). Proliferation and multi-lineage differentiation were evaluated in adipose stem cells from donor-matched ScAT and IPFP. RNA sequencing (RNA-seq) and proteomics were conducted to uncover potential molecular discrepancy in adipose stem cells and their corresponding matrix microenvironments. We found that stem cells from ScAT exhibited significantly higher proliferation and adipogenic capacity compared to those from donor-matched IPFP while stem cells from IPFP displayed significantly higher chondrogenic potential compared to those from donor-matched ScAT. Our findings are strongly endorsed by supportive data from transcriptome and proteomics analyses, indicating a site-dependent lineage preference of adipose stem cells.

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor’s characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (βThal+). For this purpose, RBC units from fourteen βThal+ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the βThal+ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the βThal+ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in βThal+ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that βThal+ erythrocytes maintain a considerable “proteo-vigilance” during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD+) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in “Glucose 6-phosphate dehydrogenase deficient subjects may be better “storers” than donors of red blood cells” [1]. Keywords: G6PD deficiency, Red blood cell storage lesion, Oxidative stress, Cell fragility, Microparticles

Although surgical and endovascular revascularization can be performed in peripheral arterial disease (PAD), 40% of patients with critical limb ischemia do not have a revascularization option. This study examines the efficacy and mechanisms of action of acellular extracellular matrix-based hydrogels as a potential novel therapy for treating PAD. We tested the efficacy of using a tissue-specific injectable hydrogel derived from decellularized porcine skeletal muscle (SKM) and compared this to a new human umbilical cord-derived matrix (hUC) hydrogel, which could have greater potential for tissue regeneration because of the younger age of the tissue source. In a rodent hindlimb ischemia model, both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis to 35 days post-injection. There were significant improvements in hindlimb tissue perfusion and perfusion kinetics with both biomaterials. Histologic analysis indicated that the injected hydrogels were biocompatible, and resulted in arteriogenesis, rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue-specific SKM hydrogel more closely matched healthy tissue morphology. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from 95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Prostate cancer (PCa) is the most common cancer in men, and the global burden of the disease is rising. The majority of PCa deaths are due to metastasis that are highly resistant to current hormonal treatments; this state is called castration-resistant prostate cancer (CRPC). In this study, we focused on the role of the lipid catabolism enzyme CPT1A in supporting CRPC growth in an androgen-dependent manner. We found that androgen withdrawal promoted the growth of CPT1A over-expressing (OE) tumors while it decreased the growth of CPT1A under-expressing (KD) tumors, increasing their sensitivity to enzalutamide. Mechanistically, we found that CPT1A-OE cells burned more lipid and showed increased histone acetylation changes that were partially reversed with a p300 specific inhibitor. Conversely, CPT1A-KD cells showed less histone acetylation when grown in androgen-deprived conditions. Our results suggest that CPT1A supports CRPC by supplying acetyl groups for histone acetylation, promoting growth and antiandrogen resistance.

Binding immunoglobulin protein (BiP) is a molecular chaperone important for the folding of numerous proteins, which include millions of immunoglobulins in human body. It also plays a key role in the unfolded protein response (UPR) in the endoplasmic reticulum. Free radical generation is a common phenomenon that occurs in cells under healthy as well as under stress conditions such as ageing, inflammation, alcohol consumption, and smoking. These free radicals attack the cell membranes and generate highly reactive lipid peroxidation products such as 4-oxononenal (4-ONE). BiP is a key protein that is modified by 4-ONE. In this study, we probed how such chemical modification affects the biophysical properties of BiP. Upon modification, BiP shows significant tertiary structural changes with no changes in its secondary structure. The protein loses its thermodynamic stability, particularly, that of the nucleotide binding domain (NBD) where ATP binds. In terms of function, the modified BiP completely loses its ATPase activity with decreased ATP binding affinity. However, modified BiP retains its immunoglobulin binding function and its chaperone activity of suppressing non-specific protein aggregation. These results indicate that 4-ONE modification can significantly affect the structure-function of key proteins such as BiP involved in cellular pathways, and provide a molecular basis for how chemical modifications can result in the failure of quality control mechanisms inside the cell.