Leucine-tRNA-synthase-2-expressing B cells contribute to colorectal cancer immunoevasion.

Immunoregulatory B cells impede antitumor immunity through unknown features and mechanisms. We report the existence of leucine-tRNA-synthase-2 (LARS2)-expressing B cell (LARS B) subset with a transforming growth factor-ß1 (TGF-ß1)-dominant regulatory feature in both mouse and human progressive colorectal cancer (CRC). Of note, LARS B cells exhibited a leucine nutrient preference and displayed active mitochondrial aminoacyl-tRNA biosynthesis. They were located outside the tertiary lymphoid structure and correlated with colorectal hyperplasia and shortened survival in CRC patients. A leucine diet induced LARS B cell generation, whereas LARS B cell deletion by Lars2 gene ablation or leucine blockage repressed CRC immunoevasion. Mechanistically, LARS2 programmed mitochondrial nicotinamide adenine dinucleotide (NAD+) regeneration and oxidative metabolism, thus determining the regulatory feature of LARS B cells in which the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) was involved. We propose a leucine-dieting scheme to inhibit LARS B cells, which is safe and useful for CRC therapy.

Cholesterol-Modified DNA Nanostructures Serve as Effective Non-Viral Carriers for Delivering siRNA to the Kidneys to Prevent Acute Kidney Injury.

With the emergence of gene therapy utilizing viral vectors, the potential risks associated with these vectors have prompted increased attention toward non-viral alternatives. DNA nanotechnology enables the assembly of specific oligonucleotide chains into nanostructures possessing defined spatial configurations. Due to their inherent characteristics, DNA nanostructures possess natural advantages as carriers for regulating gene expression in a non-viral manner. Cholesterol modification can convert DNA nanostructures from hydrophilic materials to amphiphilic materials, thereby extending their systemic circulation time. In this study, the high-dimensional design and cholesterol modification are shown to prolong the systemic circulation half-life of DNA nanostructures in mice. Specifically, the tetrahedron structure modified with three cholesterol molecules (TDN-3Chol) exhibit excellent circulation time and demonstrate a preference for renal uptake. The unique characteristics of TDN-3Chol can effectively deliver p53 siRNA to the mouse renal tubular tissue, resulting in successful knockdown of p53 and demonstrating its potential for preventing acute kidney injury. Furthermore, TDN-3Chol is not exhibited significant toxicity in mice, highlighting its promising role as a non-viral vector for targeted gene expression regulation in the kidneys. The designed non-viral vector as a prophylactic medication shows potential in addressing the current clinical challenges associated with nephrotoxic drugs.

Lab-on-Device Synthesis of Hierarchical Macro/Mesoporous WO3 Semiconducting Films for High-Performance H2S Sensing.

The performance consistency of the gas sensor is strongly dependent on the interface binding between the sensitive materials and the electrodes. Traditional powder coating methods can inevitably lead to differences in terms of substrate-film interface interaction and device performance, affecting the stability and lifetime. Thus, efficient growth of sensitive materials on device substrates is crucial and essential to enhance the sensing performance, especially for stability. Herein, hierarchically ordered macro/mesoporous WO3 films are in situ synthesized on the electrode via a facile soft/hard dual-template strategy. Orderly arrayed uniform polystyrene (PS) microspheres with tailored size (ca. 1.2 µm) are used as a hard template, and surfactant Pluronic F127 as a soft template can co-assemble with tungsten precursor into ordered mesostructure in the interstitials of PS colloidal crystal induced by solvent evaporation. Benefiting from its rich porosity and high stability, the macro/mesoporous WO3-based sensor shows high sensitivity (Rair/Rgas = 307), fast response/recovery speed (5/9 s), and excellent selectivity (SH2S/Smax > 7) toward 50 ppm H2S gas (a biomarker for halitosis). Significantly, the sensors exhibit an extended service life with a negligible change in sensing performance within 60 days. This lab-on-device synthesis provides a platform method for constructing stable nanodevices with good consistency and high stability, which are highly desired for developing high-performance sensors.

The arachidonic acid metabolome reveals elevation of prostaglandin E2 biosynthesis in colorectal cancer.

Arachidonic acid metabolites are a family of bioactive lipids derived from membrane phospholipids. They are involved in cancer progression, but arachidonic acid metabolite profiles and their related biosynthetic pathways remain uncertain in colorectal cancer (CRC). To compare the arachidonic acid metabolite profiles between CRC patients and healthy controls, quantification was performed using a liquid chromatography-mass spectrometry-based analysis of serum and tissue samples. Metabolomics analysis delineated the distinct oxidized lipids in CRC patients and healthy controls. Prostaglandin (PGE2)-derived metabolites were increased, suggesting that the PGE2 biosynthetic pathway was upregulated in CRC. The qRT-PCR and immunohistochemistry analyses showed that the expression level of PGE2 synthases, the key protein of PGE2 biosynthesis, was upregulated in CRC and positively correlated with the CD68+ macrophage density and CRC development. Our study indicates that the PGE2 biosynthetic pathway is associated with macrophage infiltration and progression of CRC tumors.

NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.

Loss of SIRT5 promotes bile acid-induced immunosuppressive microenvironment and hepatocarcinogenesis.

BACKGROUND & AIMS: The liver is a metabolically active organ and is also 'tolerogenic', exhibiting sophisticated mechanisms of immune regulation that prevent pathogen attacks and tumorigenesis. How metabolism impacts the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remains understudied. METHODS: We investigated the role of the metabolic regulator SIRT5 in HCC development by conducting metabolomic analysis, gene expression profiling, flow cytometry and immunohistochemistry analyses in oncogene-induced HCC mouse models and human HCC samples. RESULTS: We show that SIRT5 is downregulated in human primary HCC samples and that Sirt5 deficiency in mice synergizes with oncogenes to increase bile acid (BA) production, via hypersuccinylation and increased BA biosynthesis in the peroxisomes of hepatocytes. BAs act as a signaling mediator to stimulate their nuclear receptor and promote M2-like macrophage polarization, creating an immunosuppressive TME that favors tumor-initiating cells (TICs). Accordingly, high serum levels of taurocholic acid correlate with low SIRT5 expression and increased M2-like tumor-associated macrophages (TAMs) in HCC patient samples. Finally, administration of cholestyramine, a BA sequestrant and FDA-approved medication for hyperlipemia, reverses the effect of Sirt5 deficiency in promoting M2-like polarized TAMs and liver tumor growth. CONCLUSIONS: This study uncovers a novel function of SIRT5 in orchestrating BA metabolism to prevent tumor immune evasion and suppress HCC development. Our results also suggest a potential strategy of using clinically proven BA sequestrants for the treatment of patients with HCC, especially those with decreased SIRT5 and abnormally high BAs. LAY SUMMARY: Hepatocellular caricinoma (HCC) development is closely linked to metabolic dysregulation and an altered tumor microenvironment. Herein, we show that loss of the metabolic regulator Sirt5 promotes hepatocarcinogenesis, which is associated with abnormally elevated bile acids and subsequently an immunosuppressive microenvironment that favors HCC development. Targeting this mechanism could be a promising clinical strategy for HCC.

Diagnostic value of serum sphingolipids in patients with colorectal cancer.

Background: Abnormal lipid metabolism affects the regulation of tumor progression, though use of serum lipids and sphingolipids for disease progression identification is uncertain. Methods: Serum samples from 51 healthy volunteers and 76 patients were collected and analyzed by liquid chromatography tandem mass spectrometry. Results: Levels of serum total cholesterol and high-density lipoprotein were significantly lower in colorectal cancer patients. Multivariate analysis demonstrated distinct sphingolipid profiles between healthy individuals and patients. Of 106 sphingolipids, 15 metabolites that showed statistical significance were selected, and receiver operating characteristic analysis of these metabolites yielded an area under the curve of 0.868 to 0.9 by machine learning algorithms for distinguishing colorectal cancer from a healthy status. Conclusions: Healthy individuals, polyps patients and colorectal cancer patients have different serum sphingolipid signatures. Serum sphingolipids might be used as biomarkers for early detection or prediction of colorectal cancer.

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid ß-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.

Lipidomic profiling of virus infection identifies mediators that resolve herpes simplex virus-induced corneal inflammatory lesions.

Lipid mediators (LMs) play a pivotal role in the induction and resolution of inflammation. To identify and elucidate their involvement during virus infection, multiple reaction monitoring (MRM) based liquid chromatography-tandem mass spectrometry lipidomic profiling of 62 lipid species was performed in this study. Results show that RAW264.7 macrophages differentially produce specific LMs signals depending on difference in virus pathogenicity. Integration of large-scale lipidomics with targeted gene expression data revealed mediators, such as RVD3, 18-HEPE, 11(12)-EET etc. correlated with the pathogenic phase of the infection. The herpes simplex virus (HSV)-induced keratitis model demonstrates that 11(12)-EET treatment represents a novel alternative for treating viral infection.

Boronic acid-functionalized mesoporous magnetic particles with a hydrophilic surface for the multimodal enrichment of glycopeptides for glycoproteomics.

Glycosylation is an important mechanism of secondary protein processing. Large-scale profiling of glycopeptides released by proteolytic digestion of glycoproteins from biologic samples with complex compositions is limited due to their low abundance. Herein, we present a multimodal material based on boronic acid-modified mesoporous magnetic particles with a hydrophilic surface and enlarged pores around 10 nm. Multimodal enrichment successfully improved the enrichment specificity and efficiency of BMMP by synergistic interaction of hydrophilicity and boronic acid functional groups. The 10 nm pore size allows glycopeptides to enter the channel. Hydrophilic glycopeptides could be selectively enriched with an extremely low limit of detection (0.33 fmol per µL) and a high selectivity (1 : 100). From 2 µL of human serum, 328 unique glycopeptides from 101 glycoproteins were identified. A total of 33% of those glycoproteins overlapped with FDA-cleared blood serum biomarkers. It is expected that BMMP in the future can be used for large-scale biomedical glycoproteomics studies.

Deceleration of glycometabolism impedes IgG-producing B-cell-mediated tumor elimination by targeting SATB1.

B lymphocytes, known as antibody producers, mediate tumor cell destruction in the manner of antibody-dependent cell-mediated cytotoxicity; however, their anti-tumor function seems to be weakened during tumorigenesis, while the underlying mechanisms remain unclear. In this study, we found that IgG mediated anti-tumor effects, but IgG-producing B cells decreased in various tumors. Considering the underlying mechanism, glycometabolism was noteworthy. We found that tumor-infiltrating B cells were glucose-starved and accompanied by a deceleration of glycometabolism. Both inhibition of glycometabolism and deprivation of glucose through tumor cells, or glucose-free treatment, reduced the differentiation of B cells into IgG-producing cells. In this process, special AT-rich sequence-binding protein-1 (SATB1) was significantly silenced in B cells. Down-regulating SATB1 by inhibiting glycometabolism or RNA interference reduced the binding of signal transducer and activator of transcription 6 (STAT6) to the promoter of germline Cγ gene, subsequently resulting in fewer B cells producing IgG. Our findings provide the first evidence that glycometabolic inhibition by tumorigenesis suppresses differentiation of B cells into IgG-producing cells, and altering glycometabolism may be promising in improving the anti-tumor effect of B cells.

mTOR-mediated glycolysis contributes to the enhanced suppressive function of murine tumor-infiltrating monocytic myeloid-derived suppressor cells.

Immune cell activation occurs concurrently with metabolic reprogramming. As important components of the tumor microenvironment, monocytic myeloid-derived suppressor cells (M-MDSCs) are featured by their potent immunosuppressive abilities on anti-tumor effector cells. However, little is known about the contribution of metabolic adaptations to their suppressive roles. In this study, we found that tumor-infiltrating M-MDSCs had the same phenotype with splenic M-MDSCs. Compared with splenic M-MDSCs, tumor-infiltrating M-MDSCs exhibited stronger suppressive activities which was accompanied by higher glycolysis. Inhibition of glycolysis impaired the suppressive function of tumor M-MDSCs. Meanwhile, the results demonstrated that mTOR was responsible for this function regulation. mTOR inhibition by rapamycin decreased the glycolysis and reduced the suppressive activities of these cells. Furthermore, rapamycin treatment inhibited the tumor growth and reduced the percentage of M-MDSCs in 3LL tumor bearing mice. These results demonstrated that modulation of metabolism in immune cells can be an effective way to enhance anti-tumor effects.

SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense.

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose-6-phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH-producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5-dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.

Lipid metabolism in inflammation-related diseases.

There are thousands of lipid species existing in cells, which belong to eight different categories. Lipids are the essential building blocks of cells. Recent studies have started to unveil the important functions of lipids in regulating cell metabolism. However, we are still at a very early stage in fully understanding the physiological and pathological functions of lipids. The application of lipidomics for studying lipid metabolism can provide a direct readout of the cellular status and broadens our understanding of the mechanisms that underpin metabolic disease states. This review provides an introduction to lipid metabolism and its role in modulating homeostasis and immunity. We also describe representative applications of lipidomics for studying lipid metabolism in inflammation-related diseases.

Site-specific structural characterization of O-glycosylation and identification of phosphorylation sites of recombinant osteopontin.

Osteopontin (OPN) plays a key role in multiple physiological and pathological processes such as cytokine production, mineralization, inflammation, immune responses, and tumorigenesis. Post-translational modifications (PTMs) of OPN significantly affect its structure and biological properties; however, site-specific characterization of O-glycosylation in human OPN has not been reported. In this work, we profiled the overall glycan pattern of human recombinant OPN using a lectin array and completed detailed structural analysis of O-glycopeptides by mass spectrometry (MS). We detected 28 O-glycopeptides from 7 O-glycosylation regions of human OPN, occupied by highly heterogeneous O-glycans. These O-glycans carried, in part, functionally relevant epitopes such as T antigens (Galß1-3GalNAcα1-), sialyl-Tn antigens, sialyl-T antigens, and sialyl-Le(x/a) antigens [Neuα2-3Galß1-4(Fucα1-3)GlcNAc/Neuα2-3Galß1-3(Fucα1-4)GlcNAc]. MS(3) spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. Furthermore, 26 phosphorylation sites were identified by reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), including a novel one (Y209). We provide a detailed, site-specific structural characterization of O-glycosylation and identify the phosphorylation sites of OPN. These data lay the foundation for further research into the role of oligosaccharides and phosphorylation of recombinant human OPN. This article is part of a Special Issue entitled: Medical Proteomics.

Mapping and analyzing the human liver proteome: progress and potential.

INTRODUCTION: The liver is an important organ in humans. Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world. Progress in the Human Liver Proteome Project (HLPP) has improved understanding of the liver and the liver cancer proteome. AREAS COVERED: Here, we summarize the recent progress in liver proteome modification profiles, proteomic studies in liver cancer, proteomic study in the search for novel liver cancer biomarkers and drug targets, and progress of the Chromosome Centric Human Proteome Project (CHPP) in the past five years in the Institutes of Biomedical Sciences (IBS) of Fudan University. Expert commentary: Recent advances and findings discussed here provide great promise of improving the outcome of patients with liver cancer.

Transcriptome and proteome of human hepatocellular carcinoma reveal shared metastatic pathways with significant genes.

Previously isolated pathways screened from individual genes were investigated at either the transcriptional or translational level; however, the consistency between the pathways screened at the gene expression levels was obscure in metastatic human hepatocellular carcinoma (HCC). To elucidate this question, we performed a transcriptomic (16,353 genes) and proteomic (7861 proteins) analysis simultaneously on six metastatic HCC cell lines against two nonmetastatic HCC cell lines, with all HBV traceable and close genetic-backgrounds for a comparative study. The quantitative and integrated results showed that significant genes were screened differentially with 351 transcripts from the transcriptome and 304 proteins from the proteome, with limited overlapping genes (7%). However, we discovered that these discrete 351 transcripts and 304 proteins screened share extrusive significant-pathways/networks with a 77% overlap, including active TGF-ß, RAS, NFκB, and Wnt, and inactive HNF4A, which are responsible for HCC metastasis. We conclude that the discrete, but significant genes predicted by either ome play intrinsically important roles in the linkage of responsible pathways shared by both omes in HCC metastasis.

Crosstalk of homocysteinylation, methylation and acetylation on histone H3.

Homocysteine (hcy) is an intermediate metabolite in the metabolic pathway of cysteine and methionine. As a non-coded amino acid, hcy is not normally incorporated into protein. However, homocysteine can be recognized and activated by methionyl-tRNA synthetase (MetRs) to produce Hcy-thiolactone (HTL), which can react with the ε-amino group of a protein lysine residue. The N-hcy-linked protein carrying a free thiol group can influence protein structure and function, thus leading to severe diseases. Histone has multiple specific dynamic post-translational modifications (PTMs), especially on the N-terminal tail of histones enriched with lysine and arginine residues. In this study, we confirmed that histone H3 can be modified by HTL on lysine residue. Relative and absolute quantification methods based on mass spectrometry demonstrated the crosstalk between methylation and acetylation of H3 in response to excess HTL. Overall, our data provide novel insights into histone modifications and the regulatory mechanisms of diseases related to homocysteinylation.

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.