Pectin methylesterase and its proteinaceous inhibitor: a review

Pectin methylesterase (PME) catalyses the demethoxylation of pectin, a major plant cell wall polysaccharide. Through modification of the number and distribution of methyl-esters on the pectin backbone, PME affects the susceptibility of pectin towards subsequent (non-) enzymatic conversion reactions (e.g., pectin depolymerisation) and gel formation, and, hence, its functionality in both plant cell wall and pectin-containing food products. The enzyme plays a key role in vegetative and reproductive plant development in addition to plant-pathogen interactions. In addition, PME action can impact favourably or deleteriously on the structural quality of plant-derived food products. Consequently, PME and also the proteinaceous PME inhibitor (PMEI) found in several plant species and specifically inhibiting plant PMEs are highly relevant for plant biologists as well as for food technologists and are intensively studied in both fields. This review paper provides a structured, comprehensive overview of the knowledge accumulated over the years with regard to PME and PMEI. Attention is paid to both well-established and novel data concerning (i) their occurrence, polymorphism and physicochemical properties, (ii) primary and three-dimensional protein structures, (iii) catalytic and inhibitory activities, (iv) physiological roles in vivo and (v) relevance of (endogenous and exogenous) enzyme and inhibitor in the (food) industry. Remaining research challenges are indicated.     

Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.     

HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7⁺ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, β-COP. Moreover, we demonstrate that Nef contains two separable β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal α helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.     

Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.     

The Effect of Human Presence and Human Activity on Risk Assessment and Flight Initiation Distance in Skinks

Antipredator behavior and risk assessment of many species are affected by the presence of humans and their activities. Previous studies have largely been conducted on birds and mammals and relatively less is known about human impacts on reptiles. We used flight initiation distance (FID) as a measure of risk assessment in inland blue‐tailed skinks (Emoia impar) and tested the direct and indirect effects of humans on risk assessment. We first examined the effects of varying levels and types of human disturbance and activity on skink FID. We found that skinks flushed at significantly longer distances in areas with the least human activity. We then tested the degree to which skinks are able to discriminate different numbers of humans by comparing FID across three different types of approaches. Skinks did not significantly differentiate between a single approacher and a single approacher coming from a group of two other people, but did flush at greater distances when approached by three people simultaneously. Although skinks are not directly harvested or harassed by humans, they have refined human discrimination abilities. Overall, skinks habituate to a variety of human activities and perceive a larger threat when the number of human approachers is greater.     

ADP ribosylation factor 1 activity is required to recruit AP-1 to the major histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I trafficking in HIV-1-infected primary T cells

HIV-1-infected cells are partially resistant to anti-HIV cytotoxic T lymphocytes (CTLs) due to the effects of the HIV Nef protein on antigen presentation by major histocompatibility complex class I (MHC-I), and evidence has been accumulating that this function of Nef is important in vivo. HIV Nef disrupts the normal expression of MHC-I by stabilizing a protein-protein interaction between the clathrin adaptor protein AP-1 and the MHC-I cytoplasmic tail. There is also evidence that Nef activates a phosphatidylinositol 3 kinase (PI3K)-dependent GTPase, ADP ribosylation factor 6 (ARF-6), to stimulate MHC-I internalization. However, the relative importance of these two pathways is unclear. Here we report that a GTPase required for AP-1 activity (ARF-1) was needed for Nef to disrupt MHC-I surface levels, whereas no significant requirement for ARF-6 was observed in Nef-expressing T cell lines and in HIV-infected primary T cells. An ARF-1 inhibitor blocked the ability of Nef to recruit AP-1 to the MHC-I cytoplasmic tail, and a dominant active ARF-1 mutant stabilized the Nef-MHC-I-AP-1 complex. These data support a model in which Nef and ARF-1 stabilize an interaction between MHC-I and AP-1 to disrupt the presentation of HIV-1 epitopes to CTLs.     

D-phenylglycinol-derived non-covalent factor Xa inhibitors: effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity

Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.     

Evaluating butterflies as surrogates for birds and plants in semi-natural grassland buffers

Semi-natural grasslands can support diverse faunal and floral communities, including grassland birds, beneficial insects, and native wildflowers. Monitoring biodiversity of this type of ecosystem is important to assess abundance and richness of grassland-associated species, evaluate success of establishing grasslands, and to assess overall ecosystem health. We tested butterflies as surrogates for birds and plants to assess establishment success of semi-natural grassland buffers in north-central Mississippi using Spearman rank correlation (Spearman’s ρ). Disturbance and grassland butterfly guilds were generally not suitable surrogates for grassland bird metrics, non-grassland bird metrics, or nest density metrics. Butterflies did have consistent positive correlations with plant species richness and forb metrics, as well as consistent negative correlations with grass metrics, but these correlations were generally smaller than what is considered suitable to serve as surrogates. In general, butterflies were not suitable surrogates for birds or plants in semi-natural grassland buffers.