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1.  Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models 
Two ab initio modelling programs solve complementary sets of targets, enhancing the success of AMPLE with small proteins.
AMPLE clusters and truncates ab initio protein structure predictions, producing search models for molecular replacement. Here, an interesting degree of complementarity is shown between targets solved using the different ab initio modelling programs QUARK and ROSETTA. Search models derived from either program collectively solve almost all of the all-helical targets in the test set. Initial solutions produced by Phaser after only 5 min perform surprisingly well, improving the prospects for in situ structure solution by AMPLE during synchrotron visits. Taken together, the results show the potential for AMPLE to run more quickly and successfully solve more targets than previously suspected.
doi:10.1107/S1399004714025784
PMCID: PMC4321487  PMID: 25664744
AMPLE; QUARK; ROSETTA; ab initio modelling; molecular replacement
2.  Evidence for Loss of a Partial Flagellar Glycolytic Pathway during Trypanosomatid Evolution 
PLoS ONE  2014;9(7):e103026.
Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.
doi:10.1371/journal.pone.0103026
PMCID: PMC4106842  PMID: 25050549
3.  Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain 
Journal of Virology  2014;88(1):758-762.
The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle.
doi:10.1128/JVI.02294-13
PMCID: PMC3911761  PMID: 24155387
4.  Structure- and context-based analysis of the GxGYxYP family reveals a new putative class of Glycoside Hydrolase 
BMC Bioinformatics  2014;15:196.
Background
Gut microbiome metagenomics has revealed many protein families and domains found largely or exclusively in that environment. Proteins containing the GxGYxYP domain are over-represented in the gut microbiota, and are found in Polysaccharide Utilization Loci in the gut symbiont Bacteroides thetaiotaomicron, suggesting their involvement in polysaccharide metabolism, but little else is known of the function of this domain.
Results
Genomic context and domain architecture analyses support a role for the GxGYxYP domain in carbohydrate metabolism. Sparse occurrences in eukaryotes are the result of lateral gene transfer. The structure of the GxGYxYP domain-containing protein encoded by the BT2193 locus reveals two structural domains, the first composed of three divergent repeats with no recognisable homology to previously solved structures, the second a more familiar seven-stranded β/α barrel. Structure-based analyses including conservation mapping localise a presumed functional site to a cleft between the two domains of BT2193. Matching to a catalytic site template from a GH9 cellulase and other analyses point to a putative catalytic triad composed of Glu272, Asp331 and Asp333.
Conclusions
We suggest that GxGYxYP-containing proteins constitute a novel glycoside hydrolase family of as yet unknown specificity.
doi:10.1186/1471-2105-15-196
PMCID: PMC4071793  PMID: 24938123
Carbohydrate metabolism; Glycoside hydrolase; Polysaccharide Utilization Locus; PUL; Protein function prediction; JCSG; 3D structure; Protein family; Gut microbiota
5.  Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line 
Open Biology  2014;4(5):140041.
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.
doi:10.1098/rsob.140041
PMCID: PMC4042850  PMID: 24850911
titin; pseudokinase; mutagenesis; phosphorylation assay
6.  Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase 
BMC Bioinformatics  2014;15:112.
Background
Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism.
Results
BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications.
Conclusions
Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively.
doi:10.1186/1471-2105-15-112
PMCID: PMC4032388  PMID: 24742328
Glycoside hydrolase; Carbohydrate metabolism; 3D structure; Protein family; Protein function prediction; Domain of unknown function; DUF
7.  Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae 
PLoS ONE  2014;9(3):e92662.
The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae.
doi:10.1371/journal.pone.0092662
PMCID: PMC3968025  PMID: 24675797
8.  Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1 
Open Biology  2014;4(3):130172.
MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.
doi:10.1098/rsob.130172
PMCID: PMC3971405  PMID: 24671946
RBCC/TRIM fold; coiled-coil; COS-box; X-ray crystallography; electron microscopy; ab initio modelling
9.  The 2014 Nucleic Acids Research Database Issue and an updated NAR online Molecular Biology Database Collection 
Nucleic Acids Research  2013;42(Database issue):D1-D6.
The 2014 Nucleic Acids Research Database Issue includes descriptions of 58 new molecular biology databases and recent updates to 123 databases previously featured in NAR or other journals. For convenience, the issue is now divided into eight sections that reflect major subject categories. Among the highlights of this issue are six databases of the transcription factor binding sites in various organisms and updates on such popular databases as CAZy, Database of Genomic Variants (DGV), dbGaP, DrugBank, KEGG, miRBase, Pfam, Reactome, SEED, TCDB and UniProt. There is a strong block of structural databases, which includes, among others, the new RNA Bricks database, updates on PDBe, PDBsum, ArchDB, Gene3D, ModBase, Nucleic Acid Database and the recently revived iPfam database. An update on the NCBI’s MMDB describes VAST+, an improved tool for protein structure comparison. Two articles highlight the development of the Structural Classification of Proteins (SCOP) database: one describes SCOPe, which automates assignment of new structures to the existing SCOP hierarchy; the other one describes the first version of SCOP2, with its more flexible approach to classifying protein structures. This issue also includes a collection of articles on bacterial taxonomy and metagenomics, which includes updates on the List of Prokaryotic Names with Standing in Nomenclature (LPSN), Ribosomal Database Project (RDP), the Silva/LTP project and several new metagenomics resources. The NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/c/, has been expanded to 1552 databases. The entire Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/).
doi:10.1093/nar/gkt1282
PMCID: PMC3965027  PMID: 24316579
10.  Correction: Comparative Genomics of the Anopheline Glutathione S-Transferase Epsilon Cluster 
PLoS ONE  2012;7(1):10.1371/annotation/1dbf46fc-3f3f-47d0-9605-514bda135ba4.
doi:10.1371/annotation/1dbf46fc-3f3f-47d0-9605-514bda135ba4
PMCID: PMC3268786
11.  Application of the AMPLE cluster-and-truncate approach to NMR structures for molecular replacement 
Processing of NMR structures for molecular replacement by AMPLE works well.
AMPLE is a program developed for clustering and truncating ab initio protein structure predictions into search models for molecular replacement. Here, it is shown that its core cluster-and-truncate methods also work well for processing NMR ensembles into search models. Rosetta remodelling helps to extend success to NMR structures bearing low sequence identity or high structural divergence from the target protein. Potential future routes to improved performance are considered and practical, general guidelines on using AMPLE are provided.
doi:10.1107/S0907444913018453
PMCID: PMC3817692  PMID: 24189230
molecular replacement; AMPLE; NMR structures; search models
12.  The first structure in a family of peptidase inhibitors reveals an unusual Ig-like fold 
F1000Research  2013;2:154.
We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI) protein from Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig) fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in the ipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface.  Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.
doi:10.12688/f1000research.2-154.v2
PMCID: PMC3901451  PMID: 24555072
13.  The first structure in a family of peptidase inhibitors reveals an unusual Ig-like fold 
F1000Research  2013;2:154.
We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI) protein from Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig) fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in the ipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface.  Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.
doi:10.12688/f1000research.2-154.v1
PMCID: PMC3901451  PMID: 24555072
14.  Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling 
Genome Biology  2013;14(2):R11.
Background
The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan.
Results
Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms.
Conclusions
Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.
doi:10.1186/gb-2013-14-2-r11
PMCID: PMC4053784  PMID: 23375108
15.  mRNA 3′ Tagging Is Induced by Nonsense-Mediated Decay and Promotes Ribosome Dissociation 
Molecular and Cellular Biology  2012;32(13):2585-2595.
For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3′ end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3′ tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3′ tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3′ tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3′ tagging, ribosome clearance, and transcript degradation.
doi:10.1128/MCB.00316-12
PMCID: PMC3434495  PMID: 22547684
16.  Evaluating Caveolin Interactions: Do Proteins Interact with the Caveolin Scaffolding Domain through a Widespread Aromatic Residue-Rich Motif? 
PLoS ONE  2012;7(9):e44879.
Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif - a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.
doi:10.1371/journal.pone.0044879
PMCID: PMC3444507  PMID: 23028656
17.  Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions 
Molecular Biology of the Cell  2012;23(6):1095-1103.
A localization atlas is provided for 66 of 90 mammalian GFP-tagged deubiquitinases (DUBs). USP21 is the only DUB in the panel that localizes to both microtubules and the centrosome. Functional data suggest a key role for USP21 in the choreography of microtubule reorganization.
Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 deubiquitinases (DUBs) encoded in the human genome, of which 79 are predicted to have catalytic activity. We tagged 66 DUBs with green fluorescent protein and systematically surveyed their subcellular distribution, identifying enzymes specific to the nucleus, plasma membrane, and secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule-binding motif encompassed within amino acids 59–75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule- and centrosome-associated physiological processes: Depletion of USP21 in A549 cells compromises the reestablishment of a radial array of microtubules during recovery from cold-induced depolymerization and also reduces the probability of primary cilium formation, whereas USP21 knockdown in PC12 cells inhibits nerve growth factor–induced neurite outgrowth.
doi:10.1091/mbc.E11-08-0668
PMCID: PMC3302736  PMID: 22298430
18.  Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells 
Developmental Cell  2012;22-540(5):952-966.
Summary
Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions.
Graphical Abstract
Highlights
► FAM116A is a GDP-GTP exchange factor for the GTPase Rab14 ► Rab14 defines an endocytic recycling pathway required for ADAM10 transport ► In Rab14-depleted cells, ADAM10 fails to degrade its substrate, N-cadherin ► Dysregulation of ADAM10/N-cadherin accounts for Rab14 effects on cell migration
Linford et al. show that Rab14 and its exchange factor FAM116 are required for the endocytic recycling of the ADAM10 protease. In cells lacking Rab14 or FAM116, ADAM10 is mislocalized and cannot process its substrate N-cadherin. This leads to stabilization of the adherens junctions and thereby interferes with cell migration.
doi:10.1016/j.devcel.2012.04.010
PMCID: PMC3383995  PMID: 22595670
19.  Autophagy in protists 
Autophagy  2011;7(2):127-158.
Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.
doi:10.4161/auto.7.2.13310
PMCID: PMC3039767  PMID: 20962583
autophagy; ubiquitination; pexophagy; evolution; free-living protist; parasitic protist; life-cycle differentiation, Trypanosomatidae; Apicomplexa; drug discovery
20.  Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis) 
BMC Biotechnology  2011;11:85.
Background
The cotton boll weevil (Anthonomus grandis) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. In vitro directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of Bacillus thuringiensis.
Results
Bioassays carried out with A. grandis larvae revealed that the LC50 of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability.
Conclusions
The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control A. grandis.
doi:10.1186/1472-6750-11-85
PMCID: PMC3179717  PMID: 21906288
Anthonomus grandis; Bacillus thuringiensis; Cotton; DNA shuffling; Phage display; Molecular modeling
21.  Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori 
PLoS ONE  2011;6(6):e21316.
Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction.
Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.
doi:10.1371/journal.pone.0021316
PMCID: PMC3124498  PMID: 21738635
22.  New Structural and Functional Contexts of the Dx[DN]xDG Linear Motif: Insights into Evolution of Calcium-Binding Proteins 
PLoS ONE  2011;6(6):e21507.
Binding of calcium ions (Ca2+) to proteins can have profound effects on their structure and function. Common roles of calcium binding include structure stabilization and regulation of activity. It is known that diverse families – EF-hands being one of at least twelve – use a Dx[DN]xDG linear motif to bind calcium in near-identical fashion. Here, four novel structural contexts for the motif are described. Existing experimental data for one of them, a thermophilic archaeal subtilisin, demonstrate for the first time a role for Dx[DN]xDG-bound calcium in protein folding. An integrin-like embedding of the motif in the blade of a β-propeller fold – here named the calcium blade – is discovered in structures of bacterial and fungal proteins. Furthermore, sensitive database searches suggest a common origin for the calcium blade in β-propeller structures of different sizes and a pan-kingdom distribution of these proteins. Factors favouring the multiple convergent evolution of the motif appear to include its general Asp-richness, the regular spacing of the Asp residues and the fact that change of Asp into Gly and vice versa can occur though a single nucleotide change. Among the known structural contexts for the Dx[DN]xDG motif, only the calcium blade and the EF-hand are currently found intracellularly in large numbers, perhaps because the higher extracellular concentration of Ca2+ allows for easier fixing of newly evolved motifs that have acquired useful functions. The analysis presented here will inform ongoing efforts toward prediction of similar calcium-binding motifs from sequence information alone.
doi:10.1371/journal.pone.0021507
PMCID: PMC3123361  PMID: 21720552
23.  Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors 
The Journal of Cell Biology  2010;191(2):367-381.
Target or substrate Rab GTPases are identified for 17 proteins with DENN domains.
A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.
doi:10.1083/jcb.201008051
PMCID: PMC2958468  PMID: 20937701
24.  CUCU Modification of mRNA Promotes Decapping and Transcript Degradation in Aspergillus nidulans▿ † 
Molecular and Cellular Biology  2009;30(2):460-469.
In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3′ modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.
doi:10.1128/MCB.00997-09
PMCID: PMC2798463  PMID: 19901075
25.  Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure 
BMC Genomics  2009;10:611.
Background
Aspartic proteases are known to play an important role in the biology of nematode parasitism. This role is best characterised in blood-feeding nematodes, where they digest haemoglobin, but they are also likely to play important roles in the biology of nematode parasites that do not feed on blood. In the present work, we investigate the evolution and expression of aspartic proteases in Strongyloides ratti, which permits a unique comparison between parasitic and free-living adult forms within its life-cycle.
Results
We identified eight transcribed aspartic protease sequences and a further two genomic sequences and compared these to homologues in Caenorhabditis elegans and other nematode species. Phylogenetic analysis demonstrated a complex pattern of gene evolution, such that some S. ratti sequences had a one-to-one correspondence with orthologues of C. elegans but that lineage-specific expansions have occurred for other aspartic proteases in these two nematodes. These gene duplication events may have contributed to the adaptation of the two species to their different lifestyles. Among the set of S. ratti aspartic proteases were two closely-related isoforms that showed differential expression during different life stages: ASP-2A is highly expressed in parasitic females while ASP-2B is predominantly found in free-living adults. Molecular modelling of the ASP-2 isoforms reveals that their substrate specificities are likely to be very similar, but that ASP-2B is more electrostatically negative over its entire molecular surface than ASP-2A. This characteristic may be related to different pH values of the environments in which these two isoforms operate.
Conclusions
We have demonstrated that S. ratti provides a powerful model to explore the genetic adaptations associated with parasitic versus free-living life-styles. We have discovered gene duplication of aspartic protease genes in Strongyloides and identified a pair of paralogues differentially expressed in either the parasitic or the free-living phase of the nematode life-cycle, consistent with an adaptive role for aspartic proteases in the evolution of nematode parasitism.
doi:10.1186/1471-2164-10-611
PMCID: PMC2805697  PMID: 20015380

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