David A. Bernlohr

Lipid-Binding Proteins: A Family of Fatty Acid and Retinoid Transport Proteins

Publisher Summary This chapter focuses on the structural analyses and comparisons between members of a multigene family of hydrophobic ligand-binding proteins. It discusses the structural motif, general characteristics of the binding cavity, ligand entry, and the portal hypothesis and provides a detailed comparison of intra- and extracellular lipid binding proteins with known crystal structures. The members of this family are referred as lipid-binding proteins (LBPs). This collection of proteins can be subdivided into two groups: the intracellular lipid-binding protein family (iLBP) and the extracellular lipid binding protein family (eLBP). The comparison primarily deals with the iLBP branch because this family is becoming structurally well characterized. However, the structural comparisons are extended to some members of the eLBP family because the basic structural motif used to bind hydrophobic ligands applies to both. The products of hydrolysis of the intestinal lipids, including fatty acids, cholesterol, monoglycerides, and lysophospholipids, have very low solubilities and are absorbed by biliary micelles in the gut. These micelles diffuse through the glycocalyx, which stabilizes an unstirred water layer at the surface of the enterocyte. The chapter concludes with a discussion of the results of site-directed mutagenesis studies, the thermodynamics of lipid binding, and considerations of protein stability and folding.

Oxidative stress and covalent modification of protein with bioactive aldehydes.

The term “oxidative stress” links the production of reactive oxygen species to a variety of metabolic outcomes, including insulin resistance, immune dysfunction, and inflammation. Antioxidant defense systems down-regulated due to disease and/or aging result in oxidatively modified DNA, carbohydrates, proteins, and lipids. Increased production of hydroxyl radical leads to the formation of lipid hydroperoxides that produce a family of α,β-unsaturated aldehydes. Such reactive aldehydes are subject to Michael addition reactions with the side chains of lysine, histidine, and cysteine residues, referred to as “protein carbonylation.” Although not widely appreciated, reactive lipids can accumulate to high levels in cells, resulting in extensive protein modification leading to either loss or gain of function. The use of mass spectrometric methods to identify the site and extent of protein carbonylation on a proteome-wide scale has expanded our view of how oxidative stress can regulate cellular processes.

THE MAMMALIAN FATTY ACID-BINDING PROTEIN MULTIGENE FAMILY: MOLECULAR AND GENETIC INSIGHTS INTO FUNCTION

Intracellular fatty acid-binding proteins associate with fatty acids and other hydrophobic biomolecules in an internal cavity, providing for solubilization and metabolic trafficking. Analyses of their in vivo function by molecular and genetic techniques reveal specific function(s) that fatty acid-binding proteins perform with respect to fatty acid uptake, oxidation and overall metabolic homeostasis.

The Role of Lipocalin 2 in the Regulation of Inflammation in Adipocytes and Macrophages

Adipose tissue-derived cytokines (adipokines) are associated with the development of inflammation and insulin resistance. However, which adipokine(s) mediate this linkage and the mechanisms involved during obesity is poorly understood. Through proteomics and microarray screening, we recently identified lipocalin 2 (LCN 2) as an adipokine that potentially connects obesity and its related adipose inflammation. Herein we show that the levels of LCN2 mRNA are dramatically increased in adipose tissue and liver of ob/ob mice and primary adipose cells isolated from Zucker obese rats, and thiazolidinedione administration reduces LCN2 expression. Interestingly, addition of LCN2 induces mRNA levels of peroxisome proliferator-activated receptor-gamma (PPARgamma) and adiponectin. Reducing LCN2 gene expression causes decreased expression of PPARgamma and adiponectin, slightly reducing insulin-stimulated Akt2 phosphorylation at Serine 473 in 3T3-L1 adipocytes. LCN2 administration to 3T3-L1 cells attenuated TNFalpha-effect on glucose uptake, expression of PPARgamma, insulin receptor substrate-1, and glucose transporter 4, and secretion of adiponectin and leptin. When added to macrophages, LCN2 suppressed lipopolysaccharide-induced cytokine production. Our data suggest that LCN2, as a novel autocrine and paracrine adipokine, acts as an antagonist to the effect of inflammatory molecules on inflammation and secretion of adipokines.

The Fatty Acid Transport Protein (FATP1) Is a Very Long Chain Acyl-CoA Synthetase

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249–254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464–523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.

Carbonylation of Adipose Proteins in Obesity and Insulin Resistance Identification of Adipocyte Fatty Acid-binding Protein as a Cellular Target of 4-Hydroxynonenal

Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a ∼2–3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased ∼3–4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1α-1 (eEF1α1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids ∼10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.

Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction

OBJECTIVE Peripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function. RESEARCH DESIGN AND METHODS GSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis. RESULTS GSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-α treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion. CONCLUSIONS These results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1.

Fatty acid transport protein 1 (FATP1) is an ∼63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16–24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.

AMP Activated Protein Kinase-α2 Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction in Mice

AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKα2 protects the heart against systolic overload–induced ventricular hypertrophy and dysfunction, we studied the response of AMPKα2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKα2 KO had no effect on ventricular structure or function under control conditions, AMPKα2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKα2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKα2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinaseThr389, p-S6Ser235, and p-eukaryotic initiation factor 4ESer209. AMPKα2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-AktSer473. These data indicate that AMPKα2 exerts a cardiac protective effect against pressure-overload–induced ventricular hypertrophy and dysfunction.

Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase.

FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids. We cloned and characterized the murine FATP4 gene and its cDNA. From database analysis we identified the human FATP4 genomic sequence. The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene. The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids. Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene. The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3%. Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart. Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids.