P.E. Kolattukudy

Biopolyester membranes of plants: cutin and suberin.

Cutin, a biopolyester composed of hydroxy and epoxy fatty acids, is the barrier between the aerial parts of higher plants and their environment. Suberin a polymer containing aromatics and polyesters, functions as a barrier in underground parts, wound surfaces, and a variety of internal organs. The composition and probable structure of these polymers are discussed. The biosynthesis of the hydroxy, epoxy, and dicarboxylic acids of the polyesters from the common cellular fatty acids is elucidated. An extracellular enzyme transfers the hydroxy and epoxyacyl moieties from their coenzyme A derivatives to the growing polyester. The enzymes acting in the biodegradation of the polyesters have been isolated from fungi, pollen, and mammals and characterized. The function and possible practical implications of these polyester barriers are briefly discussed.

Polyesters in Higher Plants

Polyesters occur in higher plants as the structural component of the cuticle that covers the aerial parts of plants. This insoluble polymer, called cutin, attached to the epidermal cell walls is composed of interesterified hydroxy and hydroxy epoxy fatty acids. The most common chief monomers are 10,16-dihydroxy C16 acid, 18-hydroxy-9,10 epoxy C18 acid, and 9,10,18-trihydroxy C18 acid. These monomers are produced in the epidermal cells by omega hydroxylation, in-chain hydroxylation, epoxidation catalyzed by P450-type mixed function oxidase, and epoxide hydration. The monomer acyl groups are transferred to hydroxyl groups in the growing polymer at the extracellular location. The other type of polyester found in the plants is suberin, a polymeric material deposited in the cell walls of a layer or two of cells when a plant needs to erect a barrier as a result of physical or biological stress from the environment, or during development. Suberin is composed of aromatic domains derived from cinnamic acid, and aliphatic polyester domains derived from C16 and C18 cellular fatty acids and their elongation products. The polyesters can be hydrolyzed by pancreatic lipase and cutinase, a polyesterase produced by bacteria and fungi. Catalysis by cutinase involves the active serine catalytic triad. The major function of the polyester in plants is as a protective barrier against physical, chemical, and biological factors in the environment, including pathogens. Transcriptional regulation of cutinase gene in fungal pathogens is being elucidated at a molecular level. The polyesters present in agricultural waste may be used to produce high value polymers, and genetic engineering might be used to produce large quantities of such polymers in plants.

A Protease-Activated Pathway Underlying Th Cell Type 2 Activation and Allergic Lung Disease

The respiratory allergens that induce experimental Th cell type 2-dependent allergic lung inflammation may be grouped into two functional classes. One class of allergens, in this study termed type I, requires priming with adjuvants remote from the lung to overcome airway tolerogenic mechanisms that ordinarily preclude allergic responses to inhaled Ags. In contrast, the other, or type II, allergen class requires neither remote priming nor additional adjuvants to overcome airway tolerance and elicit robust allergic lung disease. In this study, we show in an experimental model that diverse type II allergens share in common proteolytic activity that is both necessary and sufficient for overcoming airway tolerance and induction of pulmonary allergic disease. Inactivated protease and protease-free Ag fragments showed no allergenic potency, demonstrating that only active protease acting on endogenous substrates was essential. Furthermore, induction of airway tolerance could be aborted and allergic lung disease established by simply adding purified protease to a type I allergen. Thus, exogenous proteases are common to type II allergens and may be generally required to overcome the innate resistance of the airway to Th cell type 2 activation and allergic inflammation, raising concern for their potential contribution to diseases such as asthma.

Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in Mycobacterium tuberculosis as It Goes into a Dormancy-Like State in Culture

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the hosts immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the hosts immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.

Contribution of Monocytes/Macrophages to Compensatory Neovascularization: The Drilling of Metalloelastase-Positive Tunnels in Ischemic Myocardium

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1–negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2′-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with &bgr;-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Cutin, Suberin, and Waxes

Publisher Summary The polymers are embedded in or associated with a complex mixture of relatively nonpolar lipids that are collectively called wax because of the similarity of their physical properties to those of the honeycomb material. In most plants, wax can be found on the surface and the crystalline structure of this wax is a rather unique characteristic of each species. The most widespread site of occurrence of wax is the cuticle, and the abundance of production of cuticular waxes by some aerial parts of plants allows easy removal of the most familiar and widely utilized plant waxes such as carnauba wax. Internal organs usually contain little wax, except in rare plants such as jojoba in which large amounts of wax esters are stored as the major energy reserve. This chapter presents a methodology that is useful for examining waxes and polymerized lipids. It discusses the types of compounds found in plant waxes and in lipid-derived polymers and explains their biosynthesis, degradation, and possible functions.

Transgenic Arabidopsis Plants Expressing a Fungal Cutinase Show Alterations in the Structure and Properties of the Cuticle and Postgenital Organ Fusions

A major structural component of the cuticle of plants is cutin. Analysis of the function of cutin in vivo has been limited because no mutants with specific defects in cutin have been characterized. Therefore, transgenic Arabidopsis plants were generated that express and secrete a cutinase from Fusarium solani f sp pisi. Arabidopsis plants expressing the cutinase in the extracellular space showed an altered ultrastructure of the cuticle and an enhanced permeability of the cuticle to solutes. In addition, pollen could germinate on fully differentiated leaves of cutinase-expressing plants but not on control leaves. These differences coincided with strong postgenital organ fusions. The junctions of the fusions contained pectic polysaccharides. As fused organs grew apart from each other, organ deformations and protrusions of epidermal cells developed at positions with high mechanical stress. These results demonstrate that an intact cutin layer not only is important for plant–environment interactions but also prevents fusions between different plant organs and is therefore necessary for normal epidermal differentiation and organ formation.

A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a Km of 7.57 mm and Vmax of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 °C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.

Monocyte Chemoattractant Protein-1 Induces a Novel Transcription Factor That Causes Cardiac Myocyte Apoptosis and Ventricular Dysfunction

Monocyte chemoattractant protein-1 (MCP-1; CCL2)–mediated inflammation plays a critical role in the development of ischemic heart disease (IHD). However, the gene expression changes caused by signal transduction, triggered by MCP-1 binding to its receptor CCR2, and their possible role in the development of IHD are not understood. We present evidence that MCP-1 binding to CCR2 induces a novel transcription factor (MCP-induced protein [MCPIP]) that causes cell death. Gene microarray analysis showed that when expressed in hiuman embryonic kidney 293 cells, MCPIP induced apoptotic gene families before causing cell death. Mutagenesis studies showed that the structural features required for transcription factor–like activity were also required for causing cell death. Activation of caspase-3 was detected after MCPIP transfection and Z-VAD-fmk partially inhibited cell death. Cardiomyocyte-targeted expression of MCP-1 in mice caused death by heart failure at 6 months of age. MCPIP expression increased in parallel with the development of ventricular dysfunction. In situ hybridization showed the presence of MCPIP transcripts in the cardiomyocytes and immunohistochemistry showed that MCPIP was associated with the cardiomyocyte nuclei of apoptotic cardiomyocytes. CCR2 expression in cardiomyocytes increased with the development of IHD. MCPIP production induced by MCP-1 binding to CCR2 in the cardiomyocytes is probably involved in the development of IHD in this murine model. MCPIP transcript levels were much higher in the explanted human hearts with IHD than with nonischemic heart disease. These results provide a molecular insight into how chronic inflammation and exposure to MCP-1 contributes to heart failure and suggest that MCPIP could be a potential target for therapeutic intervention.

Chemical signals from avocado surface wax trigger germination and appressorium formation in Colletotrichum gloeosporioides

The surface wax of the host, avocado (Persea americana) fruit, induced germination and appressorium formation in the spores of Colletotrichum gloeosporioides. Waxes from nonhost plants did not induce appressorium formation in this fungus, and avocado wax did not induce appressorium formation in most Colletotrichum species that infect other hosts. Bioassays of the thin-layer chromatographic fractions of the avocado wax showed that the fatty alcohol fraction was the main appressorium-inducing component. Testing of authentic n-C8 to n-C32 fatty alcohols revealed that C24 and longer-chain alcohols induced appressorium formation. Gas-liquid chromatography/mass spectrometry analysis of free fatty alcohols revealed that avocado wax contains a high content of very long chains. Waxes from nonhost plants containing an even higher content of the very long-chain alcohols did not induce appressorium formation. Waxes from nonhost plants strongly inhibited appressorium induction by avocado wax. Thus, a favorable balance between appressorium-inducing very long-chain fatty alcohols and the absence of inhibitors allows the fungus to use the host surface wax to trigger germination and differentiation of infection structures in the pathogen.