Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

Obesity and Atherogenic Dyslipidemia

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Cell membranes contain several classes of glycerophospholipids, which have numerous structural and functional roles in the cells. Polyunsaturated fatty acids, including arachidonic acid and eicosapentaenoic acid, are located at the sn-2 (but not sn-1)-position of glycerophospholipids in an asymmetrical manner. Using acyl-CoAs as donors, glycerophospholipids are formed by a de novo pathway (Kennedy pathway) and modified by a remodeling pathway (Lands' cycle) to generate membrane asymmetry and diversity. Both pathways were reported in the 1950s. Whereas enzymes involved in the Kennedy pathway have been well characterized, including enzymes in the 1-acylglycerol-3-phosphate O-acyltransferase family, little is known about enzymes involved in the Lands' cycle. Recently, several laboratories, including ours, isolated enzymes working in the remodeling pathway. These enzymes were discovered not only in the 1-acylglycerol-3-phosphate O-acyltransferase family but also in the membrane-bound O-acyltransferase family. In this review, we summarize recent studies on cloning and characterization of lysophospholipid acyltransferases that contribute to membrane asymmetry and diversity.

Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Non-alcoholic steatohepatitis: The role of oxidized low-density lipoproteins

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET-bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single gx and gz values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.

The human immunodeficiency virus (HIV) Vpr protein plays a critical role in AIDS pathogenesis, especially by allowing viral replication within nondividing cells such as mononuclear phagocytes. Most of the data obtained so far have been in experiments with endogenous Vpr protein; therefore the effects of extracellular Vpr protein remain largely unknown. We used synthetic Vpr protein to activate nuclear transcription factors activator protein-1 (AP-1) and NF-kappaB in the promonocytic cell line U937 and in primary macrophages. Synthetic HIV-1 Vpr protein activated AP-1, c-Jun N-terminal kinase, and MKK7 in both U937 cells and primary macrophages. Synthetic Vpr activated NF-kappaB in primary macrophages and to a lesser extent in U937 cells. Because synthetic Vpr activated AP-1 and NF-kappaB, which bind to the HIV-1 long terminal repeat, we investigated the effect of synthetic Vpr on HIV-1 replication. We observed that synthetic Vpr stimulated HIV-1 long terminal repeat in U937 cells and enhanced viral replication in chronically infected U1 promonocytic cells. Similarly, synthetic Vpr stimulated HIV-1 replication in acutely infected primary macrophages. Activation of transcription factors and enhancement of viral replication in U937 cells and primary macrophages were mediated by both the N-terminal and the C-terminal moieties of synthetic Vpr. Therefore, our results suggest that extracellular Vpr could fuel the progression of AIDS via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.

The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations