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Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid-binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs., Competing Interests: The authors have declared that no competing interests exist.

Protein engineering has enabled the design of molecular scaffolds that display a wide variety of sizes, shapes, symmetries and subunit compositions. Symmetric protein-based nanoparticles that display multiple protein domains can exhibit enhanced functional properties due to increased avidity and improved solution behavior and stability. Here we describe the creation and characterization of a computationally designed circular tandem repeat protein (cTRP) composed of 24 identical repeated motifs, which can display a variety of functional protein domains (cargo) at defined positions around its periphery. We demonstrate that cTRP nanoparticles can self-assemble from smaller individual subunits, can be produced from prokaryotic and human expression platforms, can employ a variety of cargo attachment strategies and can be used for applications (such as T-cell culture and expansion) requiring high-avidity molecular interactions on the cell surface.

The wine yeast Saccharomyces cerevisiae forms an integral part of wine production by converting relatively 'neutral' flavoured grape must into varietal aromatic wines. Additionally, non-Saccharomyces strains can be used with S. cerevisiae for the production of wines with more complexity. Yeast strains, to varying extents, produce and/or mediate the release of a whole range of key metabolites, which in turn contribute to enhanced aroma and flavour of the final wine, especially Sauvignon blanc. These metabolites viz. thiols are dependent on yeast-expressed enzymes during fermentation. Inoculation with an appropriate yeast will, therefore, lead to more commercial wine sales due to resultant wines with sought-after aroma and flavour. Likewise, inoculation with the incorrect yeast will have a negative effect on sales. It is also important to have quality control measures in place to ensure that the inoculated yeast strain quickly dominate, and is present throughout the fermentation process. Traditionally, the laborious contour clamped homogeneous electric field (CHEF) DNA karyotyping technique was shown to be reliable in this regard, however cutting-edge matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) biotyping is proving to be a faster alternative. As both methods have advantages and disadvantages, they should be used in complementary as opposed to competitively. Standard chemical and descriptive sensory analyses of wine also serve as evaluation and/or characterisation tools of yeast starter cultures. Additionally, metabolomic and proteomic profiling using gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of final wines was shown to be instrumental yeast evaluation tools. Therefore, this review highlights the importance and practicality of a more inclusive approach to evaluate and characterise novel yeast strains used for winemaking by deploying traditional and modern chemical and organoleptic evaluation techniques of wines in conjunction with cutting-edge omics approaches towards enhancing white wine varietal aroma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli . The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola . We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes., Competing Interests: KF, IB, DS, KJ, KD, BS, IF, BK, HM No competing interests declared, (© 2019, Forsberg et al.)

LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

LAGLIDADG homing endonucleases ("meganucleases") are highly specific DNA cleaving enzymes that are used for genome engineering. Like other enzymes that act on DNA targets, meganucleases often display binding affinities and cleavage activities that are dominated by one protein domain. To decipher the underlying mechanism of asymmetric DNA recognition and catalysis, we identified and characterized a new monomeric meganuclease (I-SmaMI), which belongs to a superfamily of homologous enzymes that recognize divergent DNA sequences. We solved a series of crystal structures of the enzyme-DNA complex representing a progression of sequential reaction states, and we compared the structural rearrangements and surface potential distributions within each protein domain against their relative contribution to binding affinity. We then determined the effects of equivalent point mutations in each of the two enzyme active sites to determine whether asymmetry in DNA recognition is translated into corresponding asymmetry in DNA cleavage activity. These experiments demonstrate the structural basis for "dominance" by one protein domain over the other and provide insights into this enzyme's conformational switch from a nonspecific search mode to a more specific recognition mode., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Sustainable energy is the need of the 21st century, not because of the numerous environmental and political reasons but because it is necessary to human civilization's energy future. Sustainable energy is loosely grouped into renewable energy, energy conservation, and sustainable transport disciplines. In this review, we deal with the renewable energy aspect focusing on the biomass from bioenergy crops to microalgae to produce biofuels to the utilization of high-throughput omics technologies, in particular proteomics in advancing our understanding and increasing biofuel production. We look at biofuel production by plant- and algal-based sources, and the role proteomics has played therein. This article is part of a Special Issue entitled: Translational Plant Proteomics., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world's population will reach 9-12 billion people demanding a food production increase of 34-70% (FAO, 2009) from today's food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future., (© 2013 Wiley Periodicals, Inc.)

We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis.

The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC; composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific)., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.

Sporulation in the filamentous bacteria Streptomyces coelicolor is a tightly regulated process involving aerial hyphae growth, chromosome segregation, septation and spore maturation. Genetic studies have identified numerous genes that regulate sporulation, including WhiA and the sigma factor WhiG. WhiA, which has been postulated to be a transcriptional regulator, contains two regions typically associated with DNA binding: an N-terminal domain similar to LAGLIDADG homing endonucleases, and a C-terminal helix-turn-helix domain. We characterized several in vitro activities displayed by WhiA. It binds at least two sporulation-specific promoters: its own and that of parABp(2). DNA binding is primarily driven by its HTH domain, but requires full-length protein for maximum affinity. WhiA transcription is stimulated by WhiG, while the WhiA protein binds directly to WhiG (leading to inhibition of WhiG-dependent transcription). These separate activities, which resemble a possible feedback loop, may help coordinate the closely timed cessation of aerial growth and subsequent spore formation.

Roundup(®) (glyphosate formulation) is a nonselective and posts emergent herbicide used for controlling aquatic weeds and different concentrations are used in cultures around the world. The objective of this investigation was to examine the effects of Roundup(®) (glyphosate formulation) on the biochemical composition, levels of lipoperoxidation, Na(+)/K(+)ATPase activity and reproductive traits in the Hyalella castroi. Amphipods were collected in summer 2009, in the southern Brazilian highlands. In the laboratory, the animals were kept in aquariums under controlled conditions for 7 days, and after this period they were exposed to 0.36, 0.52, 1.08 and 2.16 mg/l of glyphosate for 7 days. After the period of exposure, the animals were immediately frozen for determination of glycogen, proteins, lipids, triglycerides, cholesterol, levels of lipoperoxidation, and Na(+)/K(+)ATPase activity. During each day of the cultivation reproductive traits (number of reproductive pairs, ovigerous females and eggs in the marsupium) were observed. All concentrations of Roundup(®) induced significant decreases in all biochemical parameters and Na(+)/K(+)ATPase activity, and significant increase in lipoperoxidation levels. Showing this form a potentially toxic effect at very low concentrations, this pattern of results can lead to significant changes in trophic structure of limnic environments because these amphipods are important links in food chain in these habitats.

This article describes a partnership model between a university and health care organizations for teaching graduate nursing research from a framework of evidence-based practice. Nurses from health care organizations identified topics for graduate students to search the literature and synthesize evidence for guiding nursing practice. Nurse educators mentored graduate students in conducting critical appraisals of the literature. Students learned how to search for the evidence, summarize the existing research findings, and translate the findings into practice recommendations. Through presenting and discussing their findings with key stakeholders, students learned how nurses planned to integrate the evidence into practice. Nurses used the evidence-based results to improve their practice in the two partner hospitals. The partnership stimulated action for further inquiry into best practices.

Proteins of the DUF199 family, present in all Gram-positive bacteria and best characterized by the WhiA sporulation control factor in Streptomyces coelicolor, are thought to act as genetic regulators. The crystal structure of the DUF199/WhiA protein from Thermatoga maritima demonstrates that these proteins possess a bipartite structure, in which a degenerate N-terminal LAGLIDADG homing endonuclease (LHE) scaffold is tethered to a C-terminal helix-turn-helix (HTH) domain. The LHE domain has lost those residues critical for metal binding and catalysis, and also displays an extensively altered DNA-binding surface as compared with homing endonucleases. The HTH domain most closely resembles related regions of several bacterial sigma70 factors that bind the -35 regions of bacterial promoters. The structure illustrates how an invasive element might be transformed during evolution into a larger assemblage of protein folds that can participate in the regulation of a complex biological pathway.

This study compared the effect of different diets on the biochemical composition (lipids, cholesterol, proteins and glycogen) and the levels of lipoperoxidation and activity of Na(+)/K(+)ATPase of Hyalella castroi. We also investigated some patterns of the life cycle including survival, precopulatory mating pairs and number of ovigerous females after 21 days of culture with different diets. These crustaceans live in limnetic environments of the plateau (1,200 m a.s.l.) of the state of Rio Grande do Sul, in southern Brazil. Adult animals were collected in the autumn of 2006 in São José dos Ausentes. In the laboratory, the animals were kept submerged in aquariums, separated by sex, under controlled conditions. They were fed ad libitum for 21 days with different diets, and then some were used for biochemical determinations and others for observations of reproductive aspects. Statistical analysis revealed significant differences in the responses to glycogen, total proteins, lipoperoxidation levels and Na(+)/K(+)ATPase activity in both sexes of these amphipods and total lipids and total cholesterol levels only in females. These diets changed the biochemical patterns of the animals taken from the natural environment, and allowed a high survival rate but did not improve reproduction., (2009 Wiley-Liss, Inc)

The freshwater crab Aegla platensis was used as a model to induce ovarian growth by adding different neuroregulators to a pellet food formulation. Added compounds were the dopaminergic inhibitor spiperone or the enkephalinergic inhibitor naloxone, both of them at a dose of 10(-8) mol/animal. Animals were fed on the enriched pellets twice a week. After 7 wk, the gonadosomatic index (GI) was calculated as (gonad fresh weight/body fresh weight) x 100. GI significantly increased only for those females fed on spiperone pellets, compared to a control group receiving pellets with no compound added. During the assayed period, spiperone would be reverting the arrest exerted by dopamine on the neuroendocrine stimulation of ovarian growth. On the other hand, for both spiperone and naloxone a higher GI was correlated to a higher lipid content of both gonads and/or hepatopancreas, suggesting an increased energetic demand in accordance with an active investment in reproduction.

The NKG2x/CD94 (x = A, C, E) natural killer-cell receptors perform an important role in immunosurveillance by binding to HLA-E complexes that exclusively present peptides derived from MHC class I leader sequences, thereby monitoring MHC class I expression. We have determined the crystal structure of the NKG2A/CD94/HLA-E complex at 4.4-A resolution, revealing two critical aspects of this interaction. First, the C-terminal region of the peptide, which displays the most variability among class I leader sequences, interacts entirely with CD94, the invariant component of these receptors. Second, residues 167-170 of NKG2A/C account for the approximately 6-fold-higher affinity of the inhibitory NKG2A/CD94 receptor compared to its activating NKG2C/CD94 counterpart. These residues do not contact HLA-E or peptide directly but instead form part of the heterodimer interface with CD94. An evolutionary analysis across primates reveals that whereas CD94 is evolving under purifying selection, both NKG2A and NKG2C are evolving under positive selection. Specifically, residues at the CD94 interface have evolved under positive selection, suggesting that the evolution of these genes is driven by an interaction with pathogen-derived ligands. Consistent with this possibility, we show that NKG2C/CD94, but not NKG2A/CD94, weakly but specifically binds to the CMV MHC-homologue UL18. Thus, the evolution of the NKG2x/CD94 family of receptors has likely been shaped both by the need to bind the invariant HLA-E ligand and the need to avoid subversion by pathogen-derived decoys.

The present study investigated the effects of carbofuran on the energy metabolism (levels of glycogen, total proteins, total lipids, triglycerides, and lipoperoxidation), Na+/K+ATPase activity, and reproductive parameters (formation of couples, ovigerous females, and mean number of eggs) in the freshwater amphipods Hyalella pleoacuta and Hyalella curvispina. These crustaceans live in limnetic environments of the plateau (H. pleoacuta) and coastal plain (H. curvispina) of the state of Rio Grande do Sul in southern Brazil. The animals were collected in the winter of 2006 in the Vale das Trutas (28 degrees 47'00''S-49 degrees 50'53''W) in the Municipality of São José dos Ausentes, and in Gentil Lagoon (29 degrees 56'30''S, 50 degrees 07'50''W) in the Municipality of Tramandaí. In the laboratory, the amphipods were kept submerged in aquariums under controlled conditions of photoperiod (12 h light: 12 h dark), temperature (23 degrees C+/-1), and constant oxygenation. Animals were exposed to carbofuran at a dose of 5 or 50 microg/L for a period of 7 days. At the end of this period, the animals were immediately frozen for determination of the biochemical parameters, lipoperoxidation levels (TBARS), and enzyme Na+/K+ATPase activity. During each day of culture, several reproductive parameters were observed. Statistical analysis (ANOVA) revealed that carbofuran induces significant decreases in glycogen, proteins, lipids, triglycerides, and Na+/K+ATPase, as well as a significant increase in lipoperoxidation levels. Studies of all the biochemical parameters seem to be quite promising, in order to assess and predict the effects of toxicants on non-target organisms. The results also suggest that the reproductive parameters (formation of couples, ovigerous females and mean number of eggs) may provide sensitive criteria for assessing ecotoxicological effects. Furthermore, H. pleoacuta and H. curvispina are suitable organisms for use in toxicity tests, and we suggest that they are sensitive species that could be used in monitoring studies.

The aim of this study was to analyze the seasonal variations in the metabolism of Parastacus varicosus and examine the possible relationships to its reproduction. Animals were sampled (9 h to 10 h) in each month in the Gravataí River, RS, Brazil. Haemolymph samples were collected from each crayfish in the field for determination of glucose, total proteins, total lipids, and total cholesterol. Hepatopancreas, abdominal muscle, and gonads were removed for determination of glycogen, total proteins, total lipids, and total cholesterol. ANOVA revealed significant seasonal differences in the biochemical composition of all tissues studied; when the sexes were compared these parameters did not show any significant difference in the hepatopancreas and muscle. However, in haemolymph we observed significant variation only in cholesterol and lipid levels. The results suggest that the metabolic variability is related to the stage of maturation of the gonads, in females, where the hepatopancreas and other tissue studies can store and transfer reserves to support maturation to complement the food intake. Variations in the gonadosomatic and hepatosomatic indices suggest that reproduction occurs principally in summer. As in other decapods, abiotic factors such as water temperature, oxygen content, etc. influence the intermediate metabolism.

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

Immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the Dr family of Escherichia coli adhesins, structural and adhesive functions are carried out by the same subunit. Dr adhesins have been shown to bind decay-accelerating factor (DAF), collagen IV, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). We show that genes encoding Dr adhesins from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the Dr haemagglutinin (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity to DAF, decreased sensitivity of DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification.

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.

Background: The dual specificity phosphatase Cdc14 has been shown to be a critical regulator of late mitotic events in several eukaryotes, including S. cerevisiae, S. pombe. C. elegans and H. sapiens. However, Cdc14 homologs have clearly evolved to regulate distinct cellular processes and to respond to regulatory signals important for these processes. The human paralogs hCdc14A and B are the only vertebrate Cdc14 homologues studied to date, but their functions are not well understood. Therefore, it is of great interest to examine the function Cdc14 homologs in other vertebrate species., Results: We identified two open reading frames from Xenopus laevis closely related to human Cdc14A, called XCdc14alpha and XCdc14beta, although no obvious paralog of the hCdc14B was found. To begin a functional characterization of Xcdc14alpha and XCdc14beta, we raised polyclonal antibodies against a conserved region. These antibodies stained both the nucleolus and centrosome in interphase Xenopus tissue culture cells, and the mitotic centrosomes. GFP-tagged version of XCdc14alpha localized to the nucleulus and GFP-XCdc14beta localized to the centrosome, although not exclusively. XCdc14alpha was also both meiotically and mitotically phosphorylated. Injection of antibodies raised against a conserved region of XCdc14/beta into Xenopus embryos at the two-cell stage blocked division of the injected blastomeres, suggesting that activities of XCdc14alpha/beta are required for normal cell division., Conclusion: These results provide evidence that XCdc14alpha/beta are required for normal cellular division and are regulated by at least two mechanisms, subcellular localization and possibly phosphorylation. Due to the high sequence conservation between Xcdc14alpha and hCdc14A, it seems likely that both mechanisms will contribute to regulation of Cdc14 homologs in vertebrates.

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

We show that human Cdc14A phosphatase interacts with interphase centrosomes, and that this interaction is independent of microtubules and Cdc14A phosphatase activity, but requires active nuclear export. Disrupting the nuclear export signal (NES) led to Cdc14A being localized in nucleoli, which in unperturbed cells selectively contain Cdc14B (ref. 1). Conditional overproduction of Cdc14A, but not its phosphatase-dead or NES-deficient mutants, or Cdc14B, resulted in premature centrosome splitting and formation of supernumerary mitotic spindles. In contrast, downregulation of endogenous Cdc14A by short inhibitory RNA duplexes (siRNA) induced mitotic defects including impaired centrosome separation and failure to undergo productive cytokinesis. Consequently, both overexpression and downregulation of Cdc14A caused aberrant chromosome partitioning into daughter cells. These results indicate that Cdc14A is a physiological regulator of the centrosome duplication cycle, which, when disrupted, can lead to genomic instability in mammalian cells.

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.