Brendan J. McConkey

TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis

Recently, TDP-43 was identified as a key component of ubiquitinated aggregates in amyotrophic lateral sclerosis (ALS), an adult-onset neurological disorder that leads to the degeneration of motor neurons. Here we report eight missense mutations in nine individuals—six from individuals with sporadic ALS (SALS) and three from those with familial ALS (FALS)—and a concurring increase of a smaller TDP-43 product. These findings further corroborate that TDP-43 is involved in ALS pathogenesis.

Promotion of Plant Growth by Bacterial ACC Deaminase

To date, there has been only limited commercial use of plant growth-promoting bacteria in agriculture, horticulture, and silviculture. However, with recent progress toward understanding the mechanisms that these organisms utilize to facilitate plant growth, the use of plant growth-promoting bacteria is expected to continue to increase worldwide. One of the key mechanisms employed by plant growth-promoting bacteria to facilitate plant growth is the lowering of plant ethylene levels by the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. This article reviews the published work on this enzyme, with an emphasis on its biochemistry, protein structure, genes, and regulation. In addition, this article provides some initial insights into the changes in both plants and ACC deaminase-containing plant growth-promoting bacteria as a consequence of plant-microbe interactions. Finally, a brief discussion of how bacterial ACC deaminase and indoleacetic acid (IAA) together modulate plant growth and development is included.

The origin recognition complex protein family

SummaryOrigin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.

Pathogen-secreted proteases activate a novel plant immune pathway

Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease–G-protein–RACK1–MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

Ordered surface carbons distinguish antifreeze proteins and their ice-binding regions

Antifreeze proteins (AFPs) are found in cold-adapted organisms and have the unusual ability to bind to and inhibit the growth of ice crystals. However, the underlying molecular basis of their ice-binding activity is unclear because of the difficulty of studying the AFP-ice interaction directly and the lack of a common motif, domain or fold among different AFPs. We have formulated a generic ice-binding model and incorporated it into a physicochemical pattern-recognition algorithm. It successfully recognizes ice-binding surfaces for a diverse range of AFPs, and clearly discriminates AFPs from other structures in the Protein Data Bank. The algorithm was used to identify a novel AFP from winter rye, and the antifreeze activity of this protein was subsequently confirmed. The presence of a common and distinct physicochemical pattern provides a structural basis for unifying AFPs from fish, insects and plants.

Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.

New insights into 1-aminocyclopropane-1-carboxylate (ACC) deaminase phylogeny, evolution and ecological significance.

The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth–promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganisms developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications.

Cold-Active Winter Rye Glucanases with Ice-Binding Capacity

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic β-1,3-glucanases and an acidic β-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%–35% at −4°C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye β-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one β-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.

Effects of Bacterial ACC Deaminase on Brassica napus Gene Expression

Plants in association with plant growth-promoting rhizobacteria can benefit from lower plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. This enzyme cleaves the immediate biosynthetic precursor of ethylene, ACC. Ethylene is responsible for many aspects of plant growth and development but, under stressful conditions, it exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4 is a potent plant growth-promoting strain and, as such, was used to elaborate the detailed role of bacterial ACC deaminase in Brassica napus (canola) plant growth promotion. Transcriptional changes in bacterially treated canola plants were investigated with the use of an Arabidopsis thaliana oligonucleotide microarray. A heterologous approach was necessary because there are few tools available at present to measure global expression changes in nonmodel organisms, specifically with the sensitivity of microarrays. The results indicate that the transcription of genes involved in plant hormone regulation, secondary metabolism, and stress response was altered in plants by the presence of the bacterium, whereas the upregulation of genes for auxin response factors and the downregulation of stress response genes was observed only in the presence of bacterial ACC deaminase. These results support the suggestion that there is a direct link between ethylene and the auxin response, which has been suggested from physiological studies, and provide more evidence for the stress-reducing benefits of ACC deaminase-expressing plant growth-promoting bacteria.

Apoptosis induction by eIF5A1 involves activation of the intrinsic mitochondrial pathway.

The regulatory role of eukaryotic translation initiation factor 5A1 (eIF5A1) in apoptosis was examined using HT‐29 and HeLa S3 cells. eIF5A is the only known protein to contain the unusual amino acid, hypusine, and eIF5A1 is one of two human eIF5A family members. Two observations indicated that eIF5A1 is involved in apoptosis. First, siRNA‐mediated suppression of eIF5A1 resulted in inhibition of apoptosis induced by various apoptotic stimuli, and second, adenovirus‐mediated over‐expression of eIF5A1 strongly induced apoptotic cell death. A mutant of eIF5A1 incapable of being hypusinated also induced apoptosis when over‐expressed indicating that unhypusinated eIF5A1 is the pro‐apoptotic form of the protein. Over‐expression of eIF5A1 or of the mutant resulted in loss of mitochondrial transmembrane potential, translocation of Bax to the mitochondria, release of cytochrome c, caspase activation, up‐regulation of p53, and up‐regulation of Bim, a pro‐apoptotic BH3‐only Bcl‐2 family protein. In addition, BimL and BimS, the pro‐apoptotic alternative spliced forms of Bim, were induced in response to over‐expression of eIF5A1. Thus eIF5A1 appears to induce apoptosis by activating the mitochondrial apoptotic pathway. Proteomic analyses indicated that, of 1,899 proteins detected, 131 showed significant changes in expression (P ≤ 0.05, expression ratio ≥1.5) within 72 h of eIF5A1 up‐regulation. Among these are proteins involved in translation and protein folding, transcription factors, proteins mediating proteolysis, and a variety of proteins known to be directly involved in apoptosis. These observations collectively indicate that unhypusinated eIF5A1 plays a central role in the regulation of apoptosis. J. Cell. Physiol. 223:798–809, 2010.