Oxidases,peroxidases and hydrogen peroxide: The suberin connection

Suberin is a biopolymer present in some plant cell walls that modifies their biophysical properties. It contains both poly(phenolic) and poly(aliphatic) domains that are unique and distinct in both their chemical composition and tissue and sub-cellular location. The biosynthesis of the suberin poly(phenolic) domain is hypothesized to follow a peroxidase-mediated oxidative coupling process. In order for this to work, however, there has to be a peroxidase located at the site of suberin poly(phenolic) domain assembly, as well as a source of hydrogen peroxide to enable its function. This review focuses on the involvement of peroxidases in the macromolecular assembly of the poly(phenolic) domain of suberized tissues, with particular attention to the process in solanaceous plants, (where it has been most intensively studied), and addresses the question of the origin of the hydrogen peroxide essential to it.     

The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer.

Suberized plant cell walls have three distinguishing features: (1) tissue specificity, (2) a poly(aliphatic) domain and (3) a unique, "lignin-like" poly(phenolic) domain. With respect to the latter, comparisons have often been made to lignin, but the unique phenolic composition of suberized cells yields a unique polymer better designated as a poly(phenolic) domain. Potato tubers that have been induced to suberize through wounding make an excellent model system with which the chemistry, biochemistry and macromolecular assembly of the suberin poly(phenolic) domain can be monitored. For example, wound healing potato tubers have been used to determine the unique hydroxycinnamic acid nature of its poly(phenolic) domain using specific carbon-13 labeling studies and specific chemical degradation techniques (e.g. thioacidolysis). Furthermore, a suberization-associated anionic peroxidase has been purified from suberizing potato tubers and subsequently shown to oxidize hydroxycinnamic acids (and their derivatives) in preference to monolignols, as well as yield an unique polymer in vitro. We have since extended these studies to begin analyzing the macromolecular assembly process leading to the deposition of this suberized tissue specific domain. To this end we have begun to describe an H(2)O(2)-generating system with NAD(P)H-dependent oxidase-like properties that is temporally associated with the formation of potato suberin poly(phenolics) during suberization. Herein we describe our progress to date.     

Unraveling ferulate role in suberin and periderm biology by reverse genetics

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.Key words: BAHD feruloyl acyltransferases, ferulate, periderm, potato tuber skin, suberin, suberized tissues, waxRecently published reverse genetic studies in Arabidopsis and potato identified two orthologous genes that encode a BAHD feruloyl transferase acting on aliphatics and showed that deficiency in these enzymes produces a decrease in suberin-associated ferulic acid. These results, here discussed, signify an important advance in suberin biochemistry and ultrastructure, providing a valuable new insight into the organization of the suberized tissues and their role in the control of water vapour loss.     

A suberized perimedullary reaction zone in Populus balsamifera novel for compartmentalization in trees

 Following artificial inoculation of nonhost Populus balsamifera with Ophiostoma ulmi, structural defensive tissues were formed in the xylem. Among these tissues there was a perimedullary sheath of cells, located adjacent to the invaded xylem, that originated from the dedifferentiation of perimedullary and xylem parenchyma cells. Histochemical tests revealed that this sheath was intensively suberized. A band of lignified cells was frequently detected on both sides of this suberized tissue. The formation of such a tissue at the pith margin represents a new type of anatomical barrier in relation to compartmentalization processes described for trees. Ultrastructural examination showed that the wall of cells forming this zone was generally composed of a compound middle lamella, a suberized secondary wall and a tertiary wall layer. Using colloidal gold conjugated to monoclonal antibodies against pectin and to an exoglucanase for cellulose, only limited labelling was obtained for pectin whereas labelling for cellulose was abundant in the compound middle lamella and the tertiary wall layer. In a few fibres close to this suberized zone, the latter probe also made it possible to distinguish the occasional presence of several alternating wall layers mainly composed of either suberin or cellulose. In Salix sp., another tree species belonging to the Salicaceae, this type of suberized reaction zone was also observed. The new reaction zone is similar in structure and location to a suberized barrier formed in nonwoody carnation (Dianthus caryophyllus) plants in the defense against vascular fungi. Received: 30 July 1996 / Accepted: 7 January 1997     

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

Cloning and uterus/oviduct-specific expression of a novel estrogen-regulated gene (ERG1)

The steroid hormone estrogen profoundly influences growth and differentiation programs in the reproductive tract of cycling and pregnant mamals. It is thought that estrogen exerts its cellular effects by regulating the expression of specific target genes. We utilized a messenger RNA differential display method to identify the genes whose expression is modulated by estrogen in the preimplantation rat uterus. Here we report the cloning of a novel gene (ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that ERG1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length ERG1 cDNA indicates that it has an open reading frame of 1821 nuceotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. The extracellular part of the protein contains several distinct structural motifs. These include a zona pellucida binding domain, which is present in a number of proteins such as the zona pellucida sperm binding proteins, and uromodulin, In addition, there is a repeat of a motif called CUB domain, which exists in a number of genes involved in development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its unique pattern of expression in the uterus and oviduct and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy.     

Seasonal Populations of Pratylenchus penetrans and Meloidogyne hapla in Strawberry Roots

Strawberry roots were sampled through the year to determine the populations and distribution of Pratylenchus penetrans and Meloidogyne hapla. Three strawberry root types were sampled—structural roots; feeder roots without secondary tissues; and suberized, black perennial roots. Both lesion and root-knot nematodes primarily infected feeder roots from structural roots or healthy perennial roots. Few nematodes were recovered from soil, diseased roots, or suberized roots. Lesion nematode recovery was correlated with healthy roots. In both 1997 and 1998, P. penetrans populations peaked about day 150 (end of May) and then declined. The decline in numbers corresponded to changes in total strawberry root weight and root type distribution. The loss of nematode habitat resulted from loss of roots due to disease and the transition from structural to suberized perennial roots. Meloidogyne hapla juvenile recovery peaked around 170 days (mid June) in 1997 and at 85, 147, 229, and 308 days (late March, late May, mid August, and early November, respectively) in 1998. There appear to be at least four generations per year of M. hapla in Connecticut. Diagnostic samples from an established strawberry bed may be most reliable and useful when they include feeder roots taken in late May.     

Structural aspects of agonism and antagonism in the oestrogen receptor

We have determined the three-dimensional structures of both alpha- and beta-forms of the ligand-binding domain of the oestrogen receptor (ER) in complexes with a range of receptor agonists and antagonists. Here, we summarize how these structures provide both an understanding of the ER's distinctive pharmacophore and a rationale for its ability to bind a diverse range of chemically distinct compounds. In addition, these studies provide a unique insight into the mechanisms that underlie receptor activation, as well as providing a structural basis for the antagonist action of molecules, such as raloxifene.     

Ligand and coactivator identity determines the requirement of the charge clamp for coactivation of the peroxisome proliferator-activated receptor gamma

The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.     

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.     

Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins

CD34 and podocalyxin are structurally related sialomucins, which are expressed in multiple tissues including vascular endothelium and hematopoietic progenitors. These glycoproteins have been proposed to be involved in processes as diverse as glomerular filtration, inhibition of stem cell differentiation, and leukocyte-endothelial adhesion. Using homologies present in the cytoplasmic tails of these proteins, we have identified a novel member of this family, which we designate endoglycan. This protein shares a similar overall domain structure with the other family members including a sialomucin domain, but also possesses an extremely acidic amino-terminal region. In addition, endoglycan contains several potential glycosaminoglycan attachment sites and is modified with chondroitin sulfate. Endoglycan mRNA and protein were detected in both endothelial cells and CD34(+) bone marrow cells. Thus, CD34, podocalyxin, and endoglycan comprise a family of sialomucins sharing both structural similarity and sequence homology, which are expressed by both endothelium and multipotent hematopoietic progenitors. While the members of this family may perform overlapping functions at these sites, the unique structural features of endoglycan suggest distinct functions for this molecule.     

Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-A resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix alphaD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor.     

Structural insight into poly(A) binding and catalytic mechanism of human PARN

Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.     

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.     

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.     

Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases

Previous studies in this laboratory have identified two distinct nuclear poly(A) polymerases, a 48 kDA tumor type enzyme and a 36-38 kDA liver type enzyme. To investigate the tissue and species specificity of these enzymes, nuclear extracts were prepared from various rat tissues, pig brain and two human cell lines. These as well as whole cell extract from yeast were probed for the two enzymes by immunoblot analysis using polyclonal anti-tumor poly(A) polymerase antibodies or autoimmune sera which contain antibodies specific for the liver type enzyme. Results indicate that both tumor and liver type enzymes are conserved across species ranging from rat to human. The yeast enzyme does not appear to be immunologically related to the liver or the tumor type poly(A) polymerase. The liver type enzyme appears to be specific for normal tissues whereas the tumor type enzyme is detected only in tissues in a "tumorigenic" state or cell lines originating from tumor tissues.