ABSTRACT
The polarization of leukocytes toward chemoattractants is essential for the directed migration (chemotaxis) of leukocytes. How leukocytes acquire polarity after encountering chemical gradients is not well understood. We found here that leukocyte polarity was generated by TIPE2 (TNFAIP8L2), a transfer protein for phosphoinositide second messengers. TIPE2 functioned as a local enhancer of phosphoinositide-dependent signaling and cytoskeleton remodeling, which promoted leading-edge formation. Conversely, TIPE2 acted as an inhibitor of the GTPase Rac, which promoted trailing-edge polarization. Consequently, TIPE2-deficient leukocytes were defective in polarization and chemotaxis, and TIPE2-deficient mice were resistant to leukocyte-mediated neural inflammation. Thus, the leukocyte polarizer is a dual-role phosphoinositide-transfer protein and represents a potential therapeutic target for the treatment of inflammatory diseases.
Subject(s)
Chemotaxis, Leukocyte/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Intracellular Signaling Peptides and Proteins/genetics , T-Lymphocytes/immunology , Animals , Cell Polarity/genetics , Chemotaxis, Leukocyte/physiology , Inflammation/genetics , Inflammation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositols/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , rac GTP-Binding Proteins/antagonists & inhibitorsABSTRACT
The observation that prolonged inflammation plays a causative role in cancer development has been well documented. However, an incremental process that leads from healthy to malignant phenotypes has not yet been described. Experimentally induced hepatocellular carcinoma is considered one of the representative laboratory models for studying this process. Hepatic exposure to viral infection or toxic reagents leads to chronic inflammation and gradual transformation into hepatocellular carcinoma. Here we present metabolomic profiles of hepatic cells at different stages during inflammation-induced cellular transformation by N-nitrosodiethylamine. Using gas chromatography-mass spectrometry, we quantitatively assessed the changes in cellular metabolites during the transformation process in hepatitis and liver cirrhosis. Further pathway analysis of the differentially expressed metabolites showed that carbohydrate metabolism and lipid metabolism were greatly altered in hepatitis and liver cirrhosis, respectively. Additionally, the enhanced inflammation in cirrhosis was associated with a shift from carbohydrate metabolism to lipid and amino acid metabolism. Among the differentially expressed metabolites found in diseased mouse livers, d-glucose and d-mannitol showed the most significant changes, highlighting them as potential early-diagnostic biomarkers of hepatocellular carcinoma development. Taken together, these investigations into the dynamic metabolic changes that occur during the precancerous stages of hepatocellular carcinoma add to and refine understanding of how chronic inflammation ultimately leads to cancer. Furthermore, the findings set the stage for identifying metabolites that may serve as early-diagnostic indicators of these unfolding events.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Inflammation/metabolism , Liver/metabolism , Metabolomics/methods , Animals , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/genetics , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/genetics , Cluster Analysis , Diethylnitrosamine , Gene Expression Regulation, Neoplastic/drug effects , Hepatitis, Animal/chemically induced , Hepatitis, Animal/metabolism , Humans , Inflammation/genetics , Lipopolysaccharides , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Metabolome/drug effects , Mice, Inbred C57BL , Pyrrolidines , Reverse Transcriptase Polymerase Chain Reaction , ThiocarbamatesABSTRACT
TNF-α-induced protein 8 (TNFAIP8 or TIPE) is a newly described regulator of cancer and infection. However, its precise roles and mechanisms of actions are not well understood. We report in this article that TNFAIP8 regulates Listeria monocytogenes infection by controlling pathogen invasion and host cell apoptosis in a RAC1 GTPase-dependent manner. TNFAIP8-knockout mice were resistant to lethal L. monocytogenes infection and had reduced bacterial load in the liver and spleen. TNFAIP8 knockdown in murine liver HEPA1-6 cells increased apoptosis, reduced bacterial invasion into cells, and resulted in dysregulated RAC1 activation. TNFAIP8 could translocate to plasma membrane and preferentially associate with activated RAC1-GTP. The combined effect of reduced bacterial invasion and increased sensitivity to TNF-α-induced clearance likely protected the TNFAIP8-knockout mice from lethal listeriosis. Thus, by controlling bacterial invasion and the death of infected cells through RAC1, TNFAIP8 regulates the pathogenesis of L. monocytogenes infection.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , Listeria monocytogenes/immunology , Listeriosis/genetics , Listeriosis/immunology , Animals , Apoptosis Regulatory Proteins/metabolism , Disease Resistance/genetics , Disease Resistance/immunology , Guanosine Triphosphate/metabolism , Listeriosis/metabolism , Listeriosis/mortality , Mice , Mice, Knockout , Models, Biological , Protein Binding , Tumor Necrosis Factor-alpha/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
More than half of human cancers have aberrantly upregulated phosphoinositide signals; yet how phospholipid signals are controlled during tumorigenesis is not fully understood. We report here that TIPE3 (TNFAIP8L3) is the transfer protein of phosphoinositide second messengers that promote cancer. High-resolution crystal structure of TIPE3 shows a large hydrophobic cavity that is occupied by a phospholipid-like molecule. TIPE3 preferentially captures and shuttles two lipid second messengers, i.e., phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and increases their levels in the plasma membrane. Notably, human cancers have markedly upregulated TIPE3 expression. Knocking out TIPE3 diminishes tumorigenesis, whereas enforced TIPE3 expression enhances it in vivo. Thus, the function and metabolism of phosphoinositide second messengers are controlled by a specific transfer protein during tumorigenesis.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Lipids/physiology , Neoplasms/physiopathology , Second Messenger Systems , Cell Division , Cell Survival , Gene Knockdown Techniques , Humans , Intracellular Signaling Peptides and Proteins/genetics , Neoplasms/enzymology , Neoplasms/pathology , Protein Binding , Protein Kinases/metabolism , Signal TransductionABSTRACT
Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.
Subject(s)
Bacterial Proteins/metabolism , Peptide Elongation Factor Tu/metabolism , RNA, Transfer, Amino Acyl/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Hydrolysis , Kinetics , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/genetics , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Asn/chemistry , RNA, Transfer, Asn/metabolismABSTRACT
In nature, ribosomally synthesized proteins can contain at least 22 different amino acids: the 20 common amino acids as well as selenocysteine and pyrrolysine. Each of these amino acids is inserted into proteins codon-specifically via an aminoacyl-transfer RNA (aa-tRNA). In most cases, these aa-tRNAs are biosynthesized directly by a set of highly specific and accurate aminoacyl-tRNA synthetases (aaRSs). However, in some cases aaRSs with relaxed or novel substrate specificities cooperate with other enzymes to generate specific canonical and non-canonical aminoacyl-tRNAs.
