The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior

Recently, we defined a new syndromic form of X-linked mental retardation in a 4-generation family with a unique clinical phenotype characterized by mild mental retardation, choreoathetosis, and abnormal behavior (MRXS10). Linkage analysis in this family revealed a candidate region of 13.4 Mb between markers DXS1201 and DXS991 on Xp11; therefore, mutation analysis was performed by direct sequencing in most of the 135 annotated genes located in the region. The gene (HADH2) encoding L-3-hydroxyacyl-CoA dehydrogenase II displayed a sequence alteration (c.574 C-->A; p.R192R) in all patients and carrier females that was absent in unaffected male family members and could not be found in 2,500 control X chromosomes, including in those of 500 healthy males. The silent C-->A substitution is located in exon 5 and was shown by western blot to reduce the amount of HADH2 protein by 60%-70% in the patient. Quantitative in vivo and in vitro expression studies revealed a ratio of splicing transcript amounts different from those normally seen in controls. Apparently, the reduced expression of the wild-type fragment, which results in the decreased protein expression, rather than the increased amount of aberrant splicing fragments of the HADH2 gene, is pathogenic. Our data therefore strongly suggest that reduced expression of the HADH2 protein causes MRXS10, a phenotype different from that caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, which is a neurodegenerative disorder caused by missense mutations in this multifunctional protein.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Purification of clofibrate-induced carnitine acetyltransferase from rat liver mitochondria

The enzyme carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7) has been purified to homogeneity from hepatic mitochondria of clofibrate-fed rats. It is a protein of molecular weight 56 000 composed of two non-identical subunits of molecular weight 34 000 and 25 000. The enzyme is inhibited by palmityl-CoA as well as acetyl carnitine. The inhibition by fatty acyl-CoA is competitive with respect to both the substrates, carnitine and acetyl-CoA. The inhibition by acetylcarnitine is reversed by carnitine but not by acetyl-CoA.

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.

Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the selfbiotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over selfbiotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-59 AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

Insights into the Substrate Specificity of a Thioesterase Rv0098 of Mycobacterium Tuberculosis through X-ray Crystallographic and Molecular Dynamics Studies

The crystal structure of Rv0098, a long-chain fatty acyl-CoA thioesterase from Mycobacterium tuberculosis with bound dodecanoic acid at the active site provided insights into the mode of substrate binding but did not reveal the structural basis of substrate specificities of varying chain length. Molecular dynamics studies demonstrated that certain residues of the substrate binding tunnel are flexible and thus modulate the length of the tunnel. The flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. A combination of crystallographic and molecular dynamics studies thus explained the structural basis of accommodating long chain substrates by Rv0098 of M. tuberculosis.

A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygenrich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.

Carboxilesterase na adaptação do metabolismo de lipídios do pacu à hipoxia

Os peixes são vertebrados que vivem em vários habitats. Adaptações de sua fisiologia e de sua bioquímica são estudadas para entender como conseguem sobreviver aos desafios presentes em cada ambiente. A diminuição da concentração de oxigênio dissolvido na água é um fenômeno natural cíclico em águas do Pantanal e da Amazônia. A crescente poluição antrópica dos rios da Amazônia e do Pantanal pode, somada à hipoxia, oferecer ameaça à permanência de muitas espécies de peixes. Ao estudarmos a carboxilesterase (CarbE), enzima importante para a biotransformação de xenobióticos, observamos que sua atividade diminuía em plasma de pacu, um peixe típico do Pantanal, mantido em hipoxia durante 42 horas. As carboxilesterases participam de inúmeras reações químicas no organismo, incluindo uma transesterificação capaz de produzir ésteres etílicos tóxicos de ácido graxo (FAEE, fatty acid ethyl esters) a partir de etanol e ésteres de ácidos graxos. Já que a diminuição da atividade de CarbE poderia ser uma vantagem, pois que o etanol (um produto da glicólise em peixes sob hipoxia) seria menos esterificado, resolvemos saber mais sobre a bioquímica da CarbE do plasma e do fígado de pacus. Os pacus foram submetidos à hipoxia por diminuição da concentração até 0,5 mg O2/L por meio de borbulhamento de nitrogênio na água. Os animais ficaram nessas condições por 42 horas, quando então coletamos sangue e retiramos seus fígados. A atividade de CarbE ensaiada foi 50% menor no soro e 25% menor nos microssomos de fígado se comparada com a de peixes sob 6 mg O2/L. A CarbE isolada do soro dos pacus em normoxia possui massa molecular relativa de 56.000. A eletroforese em gel desnaturante com a fração purificada rendeu três bandas, mas o gel nativo apresentou só duas bandas com atividades sobre α-naftil acetato. Inferimos que mais do que uma isoforma da enzima está no plasma dos pacus. A CarbE isolada do soro não possui atividade de lipase sobre o Tween 20, mas os microssomos dos fígados dos animais em normoxia e hipoxia possuem. No entanto, a CarbE possui atividade sobre acil-CoA assim como o microssomo de fígado. A enzima pura apresenta Vmáx três vezes maior e uma KM quatro vezes maior do que a atividade presente nos microssomos. Além disso, os microssomos do fígado dos pacus em normoxia podem hidrolisar acil-CoA com o dobro da velocidade daqueles em hipoxia. A atividade clássica de CarbE do soro foi inibida por 4-HNE a 4 mM. A atividade de acil-CoA dos microssomos de fígado também foi sensível a 4-HNE a 4 mM, enquanto 2 mM de 4-HNE inibiu metade desta atividade do soro.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Peroxisomal beta-oxidation--a metabolic pathway with multiple functions.

Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.