Journal of Biological Chemistry最新文献

Profiling the secretome for biomarkers offers an attractive, minimally invasive strategy to detect and monitor cancer. Several challenges, however, must be overcome including the broad dynamic range of biomolecules in the secretome and the requirement for selective detection of tumor-associated markers. Here, we employed a metabolic glycoengineering (MGE) strategy, using 1,3,4-O-Bu₃ManNAz, an azido-tagged, bioorthogonal metabolic precursor of sialic acid, to label the glycome of pancreatic near-normal and cancer cells to improve conventional LC-MS/MS proteomics-based biomarker discovery. By using this "MGE-LC-MS/MS" approach that incorporates MGE-enrichment into conventional LC-MS/MS proteomics, we identified several unique proteins from the secretomes of cancer cells evaluated in vitro. In addition to proteins known to be secreted, we identified several putatively intracellular, non-N-glycosylated proteins such β-glucocerebrosidase and paladin linked to pancreatic cancer (PC) as well as proteins associated with extracellular vesicles (EV) in PC such as DCTPP1. The identification of EV-associated proteins was consistent with our discovery that ManNAc analogs used in the MGE-LC-MS/MS workflow enhance EV production, creating a more complete secretome profile of PC cells. Pointing towards clinical relevance, we used MGE-LC-MS/MS to enrich PC-derived glycoproteins from plasma harvested from mice bearing xenografted human pancreatic tumors, unambiguously demonstrating that this approach can interrogate the secretomes of cancer cells for biomarker discovery. Finally, we discovered that MGE dramatically improved the production of EVs, which both aids in biomarker discovery (this study) and holds potential to facilitate biomanufacturing of these nascent drugs.

Tristetraprolin (TTP), which encodes an RNA-binding protein, was identified as a biomarker in three types of IgE-driven allergic tissues. Remarkably, in the nasal mucosa of the ragweed pollen-induced AR mouse model, TTP mRNA levels were increased approximately threefold. TTP overexpression in AR mice alleviated nasal inflammation and epithelial barrier damage, accompanied by reduced frequency of nasal spray and nasal friction, eosinophils/neutrophils/macrophages/goblet cells infiltration, and Th2 cytokines interleukin (IL)-4, IL-5, and IL-13 secretion. The impact of TTP on the activation and differentiation of Th2 cells was assessed by utilizing naïve CD4 T cells isolated from mice. We found that TTP significantly suppressed Th2 activation and differentiation, as evidenced by the decreased levels of cytokines and the percentage of Th2. Transcriptomic profiling of CD4+ T cells (with/without TTP overexpression) was analyzed, and 14 down-regulated genes containing AU-rich elements (AREs) were obtained. The study concentrated on downregulated E3 ubiquitin ligase tripartite motif 18 (TRIM18) in TTP-overexpressed CD4+ T cells. Specifically, TTP protein bound to the ARE located at positions +3640 to +3644 (5'-UAUUU-3') within the 3'UTR of mouse TRIM18, and this interaction reduces TRIM18 mRNA stability, a process that depends on the active-site residue Cys-139 within the second CCCH-type zinc finger motif of TTP. TRIM18 overexpression weakened the effects in CD4+ T cells induced by TTP overexpression. Collectively, TTP suppresses Th2 activation and differentiation in AR by modulating TRIM18 mRNA stability, highlighting their interaction as a critical pathway in allergic inflammation.

The development of therapies that boost anti-tumor immunity has transformed cancer treatment. While the efficacy of traditional therapies, such as chemotherapy and radiation therapy, is limited by toxicity and resistance, forms of immunotherapy, including immune checkpoint blockade therapies and engineered cellular therapies, have shown unprecedented success for certain patient populations. Despite these advances, therapeutic resistance remains a significant barrier, and alternative therapies are needed to overcome immune evasion mechanisms. One prominent evasive mechanism utilized by tumor cells is hypersialylation, the overexpression of glycans capped with sialic acid on the cell surface. This review focuses on the immunosuppressive role of sialic acid in cancer and highlights opportunities to target sialic acid and its binding proteins, offering a promising therapeutic perspective to counteract resistance and improve patient outcomes.

Porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine deltacoronavirus (PDCoV), cause severe watery diarrhea, vomiting, dehydration, and high mortality in piglets, leading to enormous economic losses in the swine industry worldwide. They have the capability to infect a variety of cell lines from pigs, humans, and other animals, with high risks of interspecies transmission and potential threats to public health. These viruses employ their spike glycoproteins to engage with various receptors, coreceptors, cofactors, and other host factors that further mediate membrane fusion to accomplish the entry process. This review summarizes the recent findings regarding the pathways, receptors, coreceptors, cofactors, and other host factors utilized by TGEV, PEDV, SADS-CoV, and PDCoV for cellular entry. Several important targets for antiviral therapeutics and some key aspects of the entry process for these viruses that await discovery are highlighted. A comprehensive understanding of the entry mechanisms of porcine enteric coronaviruses will provide new insight into the development of novel antiviral therapeutic strategies.

In the post-pandemic era, the persistent threat of coronaviruses demands broad-spectrum antiviral therapeutic strategies. The SARS-CoV-2 nucleocapsid protein (N protein), an essential factor for genome packaging and immune modulation, poses a promising antiviral target. Here, we determined the crystal structure of the DNA aptamer A58-T10 in complex with the N-terminal domain of the N protein (N-NTD). A58-T10 binds to the N-NTD via a unique three-tiered stem-loop that interacts with the nucleic acid-binding site of N-NTD through extensive hydrogen bonding and stacking. Structural analysis reveals that A58 contains two stem-loops with octanucleotide motifs (5'-₁₁ACCGGATT₁₉-3' and 5'-₂₆ATCGGATT₃₃-3') that specifically recognize N-NTD. Functionally, A58 inhibits N-NTD's binding to viral RNA, disrupting N protein-host cell interactions involved in immune responses. Notably, A58 exhibits broad-spectrum binding activity against N proteins from SARS-CoV-2 variants and related sarbecoviruses. These findings elucidate the specific interaction mechanism between A58 and N-NTD, highlighting its potential as an anti-sarbecovirus agent. RUNNING TITLE: Crystal structure of DNA aptamer in complex with N-NTD.

Tumor Necrosis Factor Alpha (TNF-α) is a pleiotropic cytokine that can both facilitate tumor progression and directly mediate tumor cell killing. This dual role creates a conundrum in which TNF can be either beneficial or detrimental for a tumor, depending on the context. Herein we describe the history of the cytokine, the cases in which TNF-α has been considered as a cancer immunotherapy, and the toxicities that can manifest from its use. We also add context to its activity, particularly in T cells (via the engagement of TNF receptors), as well as the epigenetic and immunoregulatory pathways that are elicited. Furthermore, we highlight the fundamental differences in the transcriptional and translational regulation of this cytokine, which plays a significant role in the context of malignancy and potential success of immunotherapies. This review aims to provide insight and background on molecular switches, cellular context, and TNF receptor dynamics that determine TNF-α's role as both tumor suppressor and promoter in different models, which is essential for deriving maximal benefit from TNF therapies.

Lipase regioselectivity (sn-1(3) vs sn-2) is crucial for synthesizing structured lipids, but the structural basis for contrasting regioselectivities in lipases with acyl-binding tunnel remains incomplete. While the correlation between a narrow tunnel entrance and sn-2 regioselectivity has been previously established, the regioselectivity of Ophiostoma piceae sterol esterase (OPE), a lipolytic enzyme with a uniquely wide tunnel entrance, remained unclear. In this study, the chiral-phase resolution of oleic species confirmed that OPE exhibits a notable sn-1(3) regioselectivity (85.8%) against trioleoylglycerol (TOG), in contrast to Candida antarctica lipase A, which possesses narrow tunnel entrance (sn-2 regioselectivity: 79.8%). Molecular dynamics simulation showed that the wide tunnel entrance of OPE facilitates stable interaction of the scissile ester group within the catalytic center only when adopting sn-1(3) binding mode; the additional C1(3)-C2 glycerol backbone linkage within TAG allows non-scissile chains to stably reside within the tunnel entrance without disrupting the interactions at the catalytic center. Overall, the results indicated that the tunnel architecture directly dictates the sn-1(3) regioselectivity of OPE, providing structural insights into the tunnel morphology-regioselectivity relationship for lipases with acyl-binding tunnel.

Scavenger receptor class B type 1 (SR-B1), best known as a high-density lipoprotein (HDL) receptor, is now recognized as a multi-ligand membrane receptor. In addition to HDL, SR-B1 binds low-density lipoprotein (LDL), hepatitis C virus, Gram-positive and Gram-negative bacteria, and inorganic silica particles. However, the mechanisms by which SR-B1 recognizes diverse ligands remain unclear. We previously reported that a basic amino acid cluster consisting of K151, K156, and K395 at the extracellular apex of SR-B1 is essential for charge-dependent binding to silica and is distinct from the known HDL-binding site. In this study, homology modeling of SR-B1 revealed the HDL-binding site is oriented toward the basic cluster. Site-directed mutagenesis demonstrated that this basic cluster is required for HDL and, to a lesser extent, LDL binding, which in turn promotes binding to silica nanoparticles. The selective binding of SR-B1 to silica nanoparticles, but not to TiO₂ nanoparticles, latex nanoparticles, monosodium urate crystals, or multiwalled carbon nanotubes, depends on specific HDL- or LDL-silica interactions. These findings suggest that HDL, and potentially LDL, may underlie SR-B1's function as a muti-ligand membrane receptor.

Bile acids (BAs), long known for roles in food emulsion, also function as biological signals. By measuring intracellular calcium, we have recently discovered that GPR39, a G protein-coupled receptor, is a receptor for 3-O-sulfated BAs including lithocholic acid 3-sulfate (LCAS), taurolithocholic acid 3-sulfate (TLCAS) and glycolithocholic acid 3-sulfate (GLCAS) in cultured cells and in pancreatic acinar cells. We have now used multiple assays from electrophysiologic recording in Xenopus oocytes, Ca²⁺ imaging, NanoBiT, ONE-GO, to TANGO to validate GPR39 activation by BAs. Among 30 BAs, only sulfated forms (LCAS, TLCAS and GLCAS) evoked GPR39 activation, activating 9 distinct Gα protein subtypes across the Gα_q, Gα_i, and Gα_12/13 subfamilies. LCAS induced phosphorylation of ERK1/2 in the pancreas and the liver, which was markedly attenuated in Gpr39 knockout mice. Mutagenesis analysis identified the key residues essential for GPR39 signaling. Our results have revealed new signaling molecules downstream of GPR39 activation.

Targeted regulation of 70 kilodalton Heat Shock Protein (HSP70) chaperones, particularly the essential cognate heat shock protein (HSC70) and its Caenorhabditis elegans ortholog (HSP-1), may hold the key to improving cellular proteostasis and ameliorating aging-associated conditions linked to protein misfolding and aggregation. However, tools to selectively alter HSP70 chaperone activity remain elusive. In this study, we pioneer the development of two novel nanobodies, B12 and H5, which specifically bind to both recombinant and endogenous HSP-1. We show that these nanobodies, differing by only two amino acids in their complementarity-determining regions, bind specifically to HSP-1 and effectively reduce both HSP-1 ATPase activity and protein folding capacity in a dose-dependent manner in vitro. We further demonstrate in vivo expression of B12, but not H5, in transgenic C. elegans strains reduces heat-stress survival and proteotoxic-stress resistance, mirroring the effects of hsp-1 knockdown via RNA interference. Our findings suggest that these nanobodies can serve as effective and specific tools for inhibiting HSP-1 chaperone activity in vivo. These discoveries provide a foundation for future research exploring the therapeutic potential of HSP70-targeting nanobodies in aging and protein misfolding diseases.