Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.Insights into many cellular processes require detailed information about interactions between the participating proteins. However, the analysis of such interactions can be challenging because of the often-diverse physicochemical properties and the abundances of the constituent proteins, as well as the sometimes wide range of affinities and complex dynamics of the interactions. One of the key challenges has been acquiring information concerning transient, low affinity interactions in highly complex cellular milieux (3, 4).Methods that allow elucidation of such information include co-localization microscopy (5), fluorescence protein Förster resonance energy transfer (4), immunoelectron microscopy (5), yeast two-hybrid (6), and affinity capture (7, 8). Among these, affinity capture (AC)¹ has the unique potential to detect all specific in vivo interactions simultaneously, including those that interact both directly and indirectly. In recent times, the efficacy of such affinity isolation experiments has been greatly enhanced through the use of sensitive modern mass spectrometric protein identification techniques (9). Nevertheless, AC suffers from several shortcomings. These include the problem of 1) distinguishing specific from nonspecific interactors (10, 11); 2) preserving and isolating all unique interactions including those that are weak and/or transient, as well as those that exchange rapidly (10, 12, 13); and 3) differentiating proximal from more distant interactions (14).We describe here an approach to address these issues, which makes use of chemical stabilization of protein assemblies in the complex cellular milieu prior to AC. Chemical stabilization is an emerging technique for stabilizing and elucidating protein associations both in vitro (15–20) and in vivo (3, 12, 14, 21–29), with mass spectrometric (MS) readout of the AC proteins and their connectivities. Such chemical stabilization methods are indeed well-established and are often used in electron microscopy for preserving complexes and subcellular structures both in the cellular milieu (3) and in purified complexes (30, 31), wherein the most reliable, stable, and established stabilization reagents is glutaraldehyde. Recently, glutaraldehyde has been applied in the “GraFix” protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30, 31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.In this work, we present a robust pipeline for determining specific protein-protein interactions and proximities from cellular milieux. The first steps of the pipeline involve the well-established techniques of flash freezing the cells of interest in liquid nitrogen and cryomilling, which have been known for over a decade (33, 34) to preserve the cellular environment, as well as having shown outstanding performance when used in analysis of macromolecular interactions in yeast (35–39), bacterial (40, 41), trypanosome (42), mouse (43), and human (44–47) systems. The resulting frozen powder, composed of intact micron chunks of cells that have great surface area and outstanding solvent accessibility, is well suited for rapid low temperature chemical stabilization using glutaraldehyde. We selected glutaraldehyde for our procedure based on the fact that it is a very reactive stabilizing reagent, even at lower temperatures, and because it has already been shown to stabilize enzymes in their functional state (48–50). We employed highly efficient, rapid, single stage affinity capture (36, 51) for isolation and bottom-up MS for analysis of the macromolecular assemblies of interest (52–54). For convenience, we have termed this approach Stabilized Affinity-Capture Mass Spectrometry (SAC-MS).     

Genetic Engineering: A Possible Strategy for Protein–Energy Malnutrition Regulation

Protein–energy malnutrition (PEM) has adversely affected the generations of developing countries. It is a syndrome that in severity causes death. PEM generally affects infants of 1–5 age group. This manifestation is maintained till adulthood in the form of poor brain and body development. The developing nations are continuously making an effort to curb PEM. However, it is still a prime concern as it was in its early years of occurrence. Transgenic crops with high protein and enhanced nutrient content have been successfully developed. Present article reviews the studies documenting genetic engineering-mediated improvement in the pulses, cereals, legumes, fruits and other crop plants in terms of nutritional value, stress tolerance, longevity and productivity. Such genetically engineered crops can be used as a possible remedial tool to eradicate PEM.     

Peptidomimetics: A Synthetic Tool for Inhibiting Protein–Protein Interactions in Cancer

International Journal of Peptide Research and Therapeutics - Protein–protein interactions (PPI) are vital in modulating biochemical pathways in many biological processes. Inhibiting PPI is a...     

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220–223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of ¹H₂O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Protein–protein interactions of huntingtin in the hippocampus

Molecular Biology - Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington’s disease (HD) results from pathological expansion of a...     

Protein Scission by Metal Ion–Ascorbate System

About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O₂ or hydrogen peroxide). Only four of them were degraded by Fe³⁺/Fe²⁺- ascorbate, twelve – by Cu²⁺/Cu⁺-ascorbate and two proteins (α- and β-caseins) were degraded by Pd²⁺ ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu²⁺/Cu⁺-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl.     

An Empirical Exploration of the (δμ)2 Genetic Distance for 213 Human Microsatellite Markers

Microsatellites are now used ubiquitously as genetic markers. One important application is to the assessment of population subdivision and phylogenetic relatedness. Such applications require a method of estimation of genetic distance. Here we examine the most widely used measure of microsatellite genetic distance, Goldstein et al.'s delta-mu squared ([delta mu]2), with respect to a large data set of 213 markers typed across samples from four diverse human populations. We find that (delta mu)2 yields plausible interpopulation distances. For the first time, we report significant interpopulation differences in mean microsatellite length, although the effect of these differences on (delta mu)2 is negligible. However, we also show that the method is extremely sensitive to one or two loci that contribute extreme values, even when a sample size of >200 loci is used. Some of these extreme loci can be removed on the grounds that some alleles carry large indels, but for others there is no clear justification for exclusion a priori. Our data suggest a rather recent African/non-African split, with an upper limit of some 70,000-80,000 years ago.     

Tree–Grass Coexistence in the Everglades Freshwater System

Mosaic freshwater landscapes exhibit tree-dominated patches —or tree islands—interspersed in a background of marshes and wet prairies. In the Florida Everglades, these patterned landscapes provide habitat for a variety of plant and animal species and are hotspots of biodiversity. Even though the emergence of patchy freshwater systems has been associated with climate histories, fluctuating hydrologic conditions, and internal feedbacks, a process-based quantitative understanding of the underlying dynamics is still missing. Here, we develop a mechanistic framework that relates the dynamics of vegetation, nutrients and soil accretion/loss through ecogeomorphic feedbacks and interactions with hydrologic drivers. We show that the stable coexistence of tree islands and marshes results as an effect of their both being (meta-) stable states of the system. However, tree islands are found to have only a limited resilience, in that changes in hydrologic conditions or vegetation cover may cause an abrupt shift to a stable marsh state. The inherent non-linear and discontinuous dynamics determining the stability and resilience of tree islands should be accounted for in efforts aiming at the management, conservation and restoration of these features.     

Mechanism for the Action of λ Exonuclease in Genetic Recombination

Lambda exonuclease degrades in vitro redundant single stranded regions which probably result in λ DNA from genetic recombination in vivo.     

Genetic Control of Mitosis: Is Protein MASTν40 an Element of the Checkpoint System?

The effect of the mast ^v40 mutation was studied using neural ganglion cells of third-instar larvae of Drosophila melanogaster. The distributions of the cells by the interphase nucleus diameter and by the distance between the sister chromosome sets in anaphase were analyzed. Three following types of defects induced by the mutation were described: (1) Monopolar mitosis or, in the case of bipolar mitosis, an abnormally short distance between the sister chromosome sets in anaphase and early telophase. We suppose that these abnormalities are caused by damage of the start and (or) motor mechanisms of centrosome separation at the beginning and in the end of mitosis. (2) Lagging and bridging of chromosomes in anaphase and early telophase. These defects seem to be related to the disruption of functioning of mitotic spindle microtubules and (or) their defective attachment to the appropriate kinetochores. (3) Unlimited division of aneuploid and polyploid cells, which may be explained either by inactivation of the checkpoint system controlling the genome ploidy or by checkpoint adaptation. Taken collectively, our results and literature data suggest that the MAST protein is an element of the checkpoint system and that division of aneuploid and polyploid cells results from inactivation of the checkpoints.     

Mutations of the Microsomal Triglyceride-Transfer–Protein Gene in Abetalipoproteinemia

Elevated plasma levels of apolipoprotein B (apoB)–containing lipoproteins constitute a major risk factor for the development of coronary heart disease. In the rare recessively inherited disorder abetalipoproteinemia (ABL) the production of apoB-containing lipoproteins is abolished, despite no abnormality of the apoB gene. In the current study we have characterized the gene encoding a microsomal triglyceride-transfer protein (MTP), localized to chromosome 4q22-24, and have identified a mutation of the MTP gene in both alleles of all individuals in a cohort of eight patients with classical ABL. Each mutant allele is predicted to encode a truncated form of MTP with a variable number of aberrant amino acids at its C-terminal end. Expression of genetically engineered forms of MTP in Cos-1 cells indicates that the C-terminal portion of MTP is necessary for triglyceride-transfer activity. Deletion of 20 amino acids from the carboxyl terminus of the 894-amino-acid protein and a missense mutation of cysteine 878 to serine both abolished activity. These results establish that defects of the MTP gene are the predominant, if not sole, cause of hereditary ABL and that an intact carboxyl terminus is necessary for activity.     

FunMod: A Cytoscape Plugin for Identifying Functional Modules in Undirected Protein–Protein Networks

The characterization of the interacting behaviors of complex biological systems is a primary objective in protein–protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein–protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies.     

Protein–Protein Interactions in DNA Base Excision Repair

The system of base excision repair (BER) ensures correction of the most abundant DNA damages in mammalian cells and plays an important role in maintaining genome stability. Enzymes and protein factors participate in the multistage BER in a coordinated fashion, which ensures repair efficiency. The suggested coordination mechanisms are based on formation of protein complexes stabilized via either direct or indirect DNA-mediated interactions. The results of investigation of direct interactions of the proteins participating in BER with each other and with other proteins are outlined in this review. The known protein partners and sites responsible for their interaction are presented for the main participants as well as quantitative characteristics of their affinity. Information on the mechanisms of regulation of protein–protein interactions mediated by DNA intermediates and posttranslational modification is presented. It can be suggested based on all available data that the multiprotein complexes are formed on chromatin independent of the DNA damage with the help of key regulators of the BER process – scaffold protein XRCC1 and poly(ADP-ribose) polymerase 1. The composition of multiprotein complexes changes dynamically depending on the DNA damage and the stage of BER process.     

Computer System “Gene Discovery” to Search for Patterns in Eukaryotic Regulatory Nucleotide Sequences

A method is proposed to automatically search for patterns in the mutual location of context signals in regulatory DNA sequences. The procedure is based on the methods of Data Mining and Knowledge Discovery software, implemented in a computer system Gene Discovery. This system was used to study erythroid-specific promoters and promoters of the endocrine-system genes from TRRD. We detected some trends in occurrence and localization of specific oligonucleotide groups.