Ligand engineering to achieve synergistic properties in a 2D bilayer supertetrahedral chalcogenide cluster-based assembled material.

Incorporating functional organic linkers into supertetrahedral chalcogenolate cluster-based materials is an effective synthetic strategy to expand structural diversity and generate tunable optical and photoelectric properties arising from synergistic effects. Herein, a mixed ligand engineering approach was adopted to design a supertetrahedral cluster-based assembled material [(Cd6Ag4(SPh)16(TPPA)(BPE)0.5)·2DMF]n (denoted as SCCAM-3) with a 2D bilayer architecture and broader visible-light absorption. Interestingly, SCCAM-3 demonstrates a long-lived afterglow at 83 K and efficient photocatalytic activity for degrading tetracycline in water.

Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement.

Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. STATEMENT OF SIGNIFICANCE: Isolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.

15-keto-PGE2 alleviates nonalcoholic steatohepatitis through its covalent modification of NF-κB factors.

15-keto-PGE2 is one of the eicosanoids with anti-inflammatory properties. In this study, we demonstrated that 15-keto-PGE2 post-translationally modified the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunits p105/p50 and p65 at Cys59 and Cys120 sites, respectively, hence inhibiting the activation of NF-κB signaling in macrophages. In mice fed a high-fat and high-sucrose diet (HFHSD), 15-keto-PGE2 treatment reduced pro-inflammatory cytokines and fasting glucose levels. In mice with non-alcoholic steatohepatitis (NASH) induced by a prolonged HFHSD, 15-keto-PGE2 treatment significantly decreased liver inflammation, lowered serum levels of alanine transaminase (ALT) and aspartate transferase (AST), and inhibited macrophage infiltration. It also reduced lipid droplet size and downregulated key regulators of lipogenesis. These findings highlight the potential of 15-keto-PGE2, through NF-κB modification, in preventing the development and progression of steatohepatitis, emphasizing the significance of endogenous lipid mediators in the inflammatory response.

A common East-Asian ALDH2 mutation causes metabolic disorders and the therapeutic effect of ALDH2 activators.

Obesity and type 2 diabetes have reached pandemic proportion. ALDH2 (acetaldehyde dehydrogenase 2, mitochondrial) is the key metabolizing enzyme of acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal. A missense Glu504Lys mutation of the ALDH2 gene is prevalent in 560 million East Asians, resulting in reduced ALDH2 enzymatic activity. We find that male Aldh2 knock-in mice mimicking human Glu504Lys mutation were prone to develop diet-induced obesity, glucose intolerance, insulin resistance, and fatty liver due to reduced adaptive thermogenesis and energy expenditure. We find reduced activity of ALDH2 of the brown adipose tissue from the male Aldh2 homozygous knock-in mice. Proteomic analyses of the brown adipose tissue from the male Aldh2 knock-in mice identifies increased 4-hydroxynonenal-adducted proteins involved in mitochondrial fatty acid oxidation and electron transport chain, leading to markedly decreased fatty acid oxidation rate and mitochondrial respiration of brown adipose tissue, which is essential for adaptive thermogenesis and energy expenditure. AD-9308 is a water-soluble, potent, and highly selective ALDH2 activator. AD-9308 treatment ameliorates diet-induced obesity and fatty liver, and improves glucose homeostasis in both male Aldh2 wild-type and knock-in mice. Our data highlight the therapeutic potential of reducing toxic aldehyde levels by activating ALDH2 for metabolic diseases.

An Atypical F-Actin Capping Protein Modulates Cytoskeleton Behaviors Crucial for Trichomonas vaginalis Colonization.

Cytoadherence and migration are crucial for pathogens to establish colonization in the host. In contrast to a nonadherent isolate of Trichomonas vaginalis, an adherent one expresses more actin-related machinery proteins with more active flagellate-amoeboid morphogenesis, amoeba migration, and cytoadherence, activities that were abrogated by an actin assembly blocker. By immunoprecipitation coupled with label-free quantitative proteomics, an F-actin capping protein (T. vaginalis F-actin capping protein subunit α [TvFACPα]) was identified from the actin-centric interactome. His-TvFACPα was detected at the barbed end of a growing F-actin filament, which inhibited elongation and possessed atypical activity in binding G-actin in in vitro assays. TvFACPα partially colocalized with F-actin at the parasite pseudopod protrusion and formed a protein complex with α-actin through its C-terminal domain. Meanwhile, TvFACPα overexpression suppressed F-actin polymerization, amoeboid morphogenesis, and cytoadherence in this parasite. Ser2 phosphorylation of TvFACPα enriched in the amoeboid stage of adhered trophozoites was reduced by a casein kinase II (CKII) inhibitor. Site-directed mutagenesis and CKII inhibitor treatment revealed that Ser2 phosphorylation acts as a switching signal to alter TvFACPα actin-binding activity and the consequent actin cytoskeleton behaviors. Through CKII signaling, TvFACPα also controls the conversion of adherent trophozoites from amoeboid migration to the flagellate form with axonemal motility. Together, CKII-dependent Ser2 phosphorylation regulates TvFACPα binding to actin to fine-tune cytoskeleton dynamics and drive crucial behaviors underlying host colonization by T. vaginalis. IMPORTANCE Trichomoniasis is one of the most prevalent nonviral sexually transmitted diseases. T. vaginalis cytoadherence to urogenital epithelium cells is the first step in the colonization of the host. However, studies on the mechanisms of cytoadherence have focused mainly on the role of adhesion molecules, and their effects are limited when analyzed by loss- or gain-of-function assays. This study proposes an extra pathway in which the actin cytoskeleton mediated by a capping protein α-subunit may play roles in parasite morphogenesis, cytoadherence, and motility, which are crucial for colonization. Once the origin of the cytoskeleton dynamics could be manipulated, the consequent activities would be controlled as well. This mechanism may provide new potential therapeutic targets to impair this parasite infection and relieve the increasing impact of drug resistance on clinical and public health.

Modulation of Thalamocingulate Nociceptive Transmission and Glutamate Secretion by Targeting P2×7 Receptor.

The complexity and diversity of pain signaling have led to obstacles for prominent treatments due to mechanisms that are not yet fully understood. Among adenosine triphosphate (ATP) receptors, P2×7 differs in many respects from P2×1-6, it plays a significant role in various inflammatory pain, but whether it plays a role in noninflammatory pain has not been widely discussed. In this study, we utilized major neuropharmacological methods to record the effects of manipulating P2×7 during nociceptive signal transmission in the thalamocingulate circuits. Our results show that regardless of the specific cell type distribution of P2×7 in the central nervous system (CNS), it participates directly in the generated nociceptive transmission, which indicates its apparent functional existence in the major pain transmission path, the thalamocingulate circuits. Activation of P2×7 may facilitate transmission velocity along the thalamocingulate projection as well as neuron firings and synaptic vesicle release in anterior cingulate cortical neurons. Targeting thalamic P2×7 affects glutamate and ATP secretion during nociceptive signal transmission. PERSPECTIVE: The observations in this study provide evidence that the ATP receptor P2×7 presents in the central ascending pain path and plays a modulatory role during nociceptive transmission, which could contribute new insights for many antinociceptive applications.

Bifunctional Supertetrahedral Chalcogenolate Cluster-Based Assembly Materials Constructed by a Photoactive Ligand.

The assembly of supertetrahedral chalcogenolate clusters (SCCs) and multifunctional organic linkers could lead to the formation of tunable structures and synergistic properties. Two SCC-based assembled materials (SCCAM-1 and -2) constructed by a triangular chromophore ligand, tris(4-pyridylphenyl)amine, were successfully synthesized and characterized. The SCCAMs demonstrate unusually long-lived afterglow at low temperatures (83 K) and efficient activities for the photocatalytic degradation of organic dye in water.

A nuclear pore complex-associated regulation of SUMOylation in meiosis.

The nuclear pore complex (NPC) provides a permeable barrier between the nucleoplasm and cytoplasm. In a subset of NPC constituents that regulate meiosis in the fission yeast Schizosaccharomyces pombe, we found that nucleoporin Nup132 (homolog of human Nup133) deficiency resulted in transient leakage of nuclear proteins during meiosis I, as observed in the nup132 gene-deleted mutant. The nuclear protein leakage accompanied the liberation of the small ubiquitin-like modifier (SUMO)-specific ubiquitin-like protease 1 (Ulp1) from the NPC. Ulp1 retention at the nuclear pore prevented nuclear protein leakage and restored normal meiosis in a mutant lacking Nup132. Furthermore, using mass spectrometry analysis, we identified DNA topoisomerase 2 (Top2) and RCC1-related protein (Pim1) as the target proteins for SUMOylation. SUMOylation levels of Top2 and Pim1 were altered in meiotic cells lacking Nup132. HyperSUMOylated Top2 increased the binding affinity at the centromeres of nup132 gene-deleted meiotic cells. The Top2-12KR sumoylation mutant was less localized to the centromeric regions. Our results suggest that SUMOylation of chromatin-binding proteins is regulated by the NPC-bound SUMO-specific protease and is important for the progression of meiosis.

Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

Deglycosylation of SLAMF7 in breast cancers enhances phagocytosis.

N-linked glycosylation of proteins is one of the post-translational modifications (PTMs) that shield tumor antigens from immune attack. Signaling lymphocytic activation molecule family 7 (SLAMF7) suppresses cancer cell phagocytosis and is an ideal target under clinical development. PTM of SLAMF7, however, remains less understood. In this study, we investigated the role of N-glycans on SLAMF7 in breast cancer progression. We identified seven N-linked glycosylation motifs on SLAMF7, which are majorly occupied by complex structures. Evolutionally conserved N98 residue is enriched with high mannose and sialylated glycans. Hyperglycosylated SLAMF7 was associated with STT3A expression in breast cancer cells. Inhibition of STT3A by a small molecule inhibitor, N-linked glycosylation inhibitor-1 (NGI-1), reduced glycosylation of SLAMF7, resulting in enhancing antibody affinity and phagocytosis. To provide an on-target effect, we developed an antibody-drug conjugate (ADC) by coupling the anti-SLAMF7 antibody with NGI-1. Deglycosylation of SLAMF7 increases antibody recognition and promotes macrophage engulfment of breast cancer cells. Our work suggests deglycosylation by ADC is a potential strategy to enhance the response of immunotherapeutic agents.

A Study on MDA5 Signaling in Splenic B Cells from an Imiquimod-Induced Lupus Mouse Model with Proteomics.

INTRODUCTION: Several environmental stimuli may influence lupus, particularly viral infections. In this study, we used an imiquimod-induced lupus mouse model focused on the TLR7 pathway and proteomics analysis to determine the specific pathway related to viral infection and the related protein expressions in splenic B cells to obtain insight into B-cell responses to viral infection in the lupus model. MATERIALS AND METHODS: We treated FVB/N wild-type mice with imiquimod for 8 weeks to induce lupus symptoms and signs, retrieved splenocytes, selected B cells, and conducted the proteomic analysis. The B cells were co-cultured with CD40L+ feeder cells for another week before performing Western blot analysis. Panther pathway analysis was used to disclose the pathways activated and the protein-protein interactome was analyzed by the STRING database in this lupus murine model. RESULTS: The lupus model was well established and well demonstrated with serology evidence and pathology proof of lupus-mimicking organ damage. Proteomics data of splenic B cells revealed that the most important activated pathways (fold enrichment > 100) demonstrated positive regulation of the MDA5 signaling pathway, negative regulation of IP-10 production, negative regulation of chemokine (C-X-C motif) ligand 2 production, and positive regulation of the RIG-I signaling pathway. A unique protein-protein interactome containing 10 genes was discovered, within which ISG15, IFIH1, IFIT1, DDX60, and DHX58 were demonstrated to be downstream effectors of MDA5 signaling. Finally, we found B-cell intracellular cytosolic proteins via Western blot experiment and continued to observe MDA5-related pathway activation. CONCLUSION: In this experiment, we confirmed that the B cells in the lupus murine model focusing on the TLR7 pathway were activated through the MDA5 signaling pathway, an important RNA sensor implicated in the detection of viral infections and autoimmunity. The MDA5 agonist/antagonist RNAs and the detailed molecular interactions within B cells are worthy of further investigation for lupus therapy.

Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness.

BACKGROUND: We recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-ß receptor enhances its signaling and promotes breast cancer invasion and metastasis. However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown. METHOD: We performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, ingenuity pathway analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. RESULTS: Loss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity pathway analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvß5 or IL6ST might be crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial-mesenchymal transition and metastasis. CONCLUSIONS: Our report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis.

Urinary Galectin-3 as a Novel Biomarker for the Prediction of Renal Fibrosis and Kidney Disease Progression.

Plasma galectin-3 (Gal-3) is associated with organ fibrosis, but whether urinary Gal-3 is a potential biomarker of kidney disease progression has never been explored. Between 2018 and 2021, we prospectively enrolled 280 patients who underwent renal biopsy and were divided into three groups based on their urinary Gal-3 levels (<354.6, 354.6−510.7, and ≥510.8 pg/mL) to assess kidney disease progression (defined as ≥40% decline in the estimated glomerular filtration rate or end-stage renal disease) and renal histology findings. Patients in the highest urinary Gal-3 tertile had the lowest eGFRs and highest proteinuria levels. In multivariate Cox regression models, patients in the highest tertile had the highest risk of kidney disease progression (adjusted hazard ratio, 4.60; 95% confidence interval, 2.85−7.71) compared to those in the lowest tertile. Higher urinary Gal-3 levels were associated with more severe renal fibrosis. Intrarenal mRNA expression of LGALS3 (Gal-3-encoded gene) was most correlated with the renal stress biomarkers (IGFBP7 and TIMB2), renal function biomarkers (PTGDS) and fibrosis-associated genes (TGFB1). The urinary Gal-3 level may be useful for the identification of patients at high risk of kidney disease progression and renal fibrosis, and for the early initiation of treatments for these patients.

Identification of Galectin-3 as Potential Biomarkers for Renal Fibrosis by RNA-Sequencing and Clinicopathologic Findings of Kidney Biopsy.

Background: Galectin-3 (Gal-3) is a multifunctional glycan-binding protein shown to be linked to chronic inflammation and fibrogenesis. Plasma Gal-3 is associated with proteinuria and renal dysfunction, but its role has never been confirmed with kidney biopsy results. In our study, we aimed to explore the expression of Gal-3 in biopsy-proven patients, and we tested the hypothesis that chronic kidney disease (CKD) leads to upregulation of plasma Gal-3 expression in corresponding biopsy findings and RNA sequencing analysis. Method: In 249 patients (male/female: 155/94, age: 57.2 ± 16.3 years) who underwent kidney biopsy, plasma levels of Gal-3 were measured to estimate the association of renal fibrosis. Relationships between plasma Gal-3 levels, estimated glomerular filtration rate (eGFR) and renal histology findings were also assessed. We further examined the gene expression of Gal-3 in RNA-sequencing analysis in biopsy-proven patients. Results: Compared to patients without CKD, CKD patients had higher levels of plasma Gal-3 (1,016.3 ± 628.1 pg/mL vs. 811.6 ± 369.6 pg/ml; P = 0.010). Plasma Gal-3 was inversely correlated with eGFR (P = 0.005) but not with proteinuria. Higher Gal-3 levels were associated with interstitial fibrosis, tubular atrophy and vascular intimal fibrosis. RNA-sequencing analysis showed the upregulation of Gal-3 in fibrotic kidney biopsy samples, and the differentially expressed genes were mainly enhanced in immune cell activation and the regulation of cell-cell adhesion. Conclusions: Plasma Gal-3 levels are inverse correlated with eGFR but positively correlated with renal fibrosis, which may be involved in the immune response and associated pathways. These findings support the role of Gal-3 as a predictive marker of renal fibrosis.

Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro.

BACKGROUND: Despite clinical success with anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines has been compromised by rapidly spreading SARS-CoV-2 variants. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host's immune response and attenuate antibody efficiency. However, it remains unclear if targeting glycosylation on viral spike protein can impair infectivity of SARS-CoV-2 and its variants. METHODS: We adopted flow cytometry, ELISA, and BioLayer interferometry approaches to assess binding of glycosylated or deglycosylated spike with ACE2. Viral entry was determined by luciferase, immunoblotting, and immunofluorescence assays. Genome-wide association study (GWAS) revealed a significant relationship between STT3A and COVID-19 severity. NF-κB/STT3A-regulated N-glycosylation was investigated by gene knockdown, chromatin immunoprecipitation, and promoter assay. We developed an antibody-drug conjugate (ADC) that couples non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) to specifically target SARS-CoV-2-infected cells. FINDINGS: The receptor binding domain and three distinct SARS-CoV-2 surface N-glycosylation sites among 57,311 spike proteins retrieved from the NCBI-Virus-database are highly evolutionarily conserved (99.67%) and are involved in ACE2 interaction. STT3A is a key glycosyltransferase catalyzing spike glycosylation and is positively correlated with COVID-19 severity. We found that inhibiting STT3A using N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 infectivity and that of its variants [Alpha (B.1.1.7) and Beta (B.1.351)]. Most importantly, 4G10-ADC enters SARS-CoV-2-infected cells and NGI-1 is subsequently released to deglycosylate spike protein, thereby reinforcing the neutralizing abilities of antibodies, vaccines, or convalescent sera and reducing SARS-CoV-2 variant infectivity. INTERPRETATION: Our results indicate that targeting evolutionarily-conserved STT3A-mediated glycosylation via an ADC can exert profound impacts on SARS-CoV-2 variant infectivity. Thus, we have identified a novel deglycosylation method suitable for eradicating SARS-CoV-2 variant infection in vitro. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation.

BACKGROUND: The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. METHODS: ApoE-deficient (apoE-/-) and apoE/Siglec-E-double deficient (apoE-/-/Siglec-E-/-) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E-/-- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays. RESULTS: Here we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE-/- mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E-/- counterparts. CONCLUSIONS: These data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.

SLC38A2 Overexpression Induces a Cancer-like Metabolic Profile and Cooperates with SLC1A5 in Pan-cancer Prognosis.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.

Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.

Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Comparison of the protein acetylome of endothelial cells upon shear flow and resveratrol treatment.

BACKGROUND: Posttranslational acetylation/deacetylation known as the acetylome is important in regulating protein activity. Shear flow (SF) and resveratrol (RSV) are two stimuli that represent physical and chemical signal separately. The acetylome co-regulated by these two stimuli remain unclear. METHODS: Human umbilical cord vein endothelial cells (HUVECs) were subjected to either SF of 12 dynes/cm² or 10 µM RSV. The purified acetylated peptides were labeled by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. The signaling cascades of the identified acetylome were predicted by ingenuity pathway analysis (IPA). Co-immunoprecipitation was applied to confirm the acetylation status of proteins. RESULTS: Five groups of proteins showed an increased acetylation upon SF and RSV treatment. After algorithm, 628 proteins with increased acetylation and 22 proteins with decreased acetylation were identified in the SF acetylome. For the acetylome regulated by RSV, 145 proteins with increased acetylation and 23 proteins with decreased acetylation were identified. Compared these two acetylomes, 129 proteins with increased acetylation and 2 proteins with decreased acetylation were co-regulated by both SF and RSV treatments. IPA analysis showed that this co-regulated acetylome was involved in heat shock response, and the signals of eNOS, STAT3, JAK/STAT and ERK/MAPK. Co-immunoprecipitation analysis further confirmed the acetylated status of mitochondrial HSP60 and mitochondrial citrate synthase. CONCLUSIONS: This study indicated that physical signal is more complicated than chemical signal in the case of acetylome. The co-regulated proteins are worthy for further study in discussing synergetic effect between physical and chemical signal in cardioprotection.