| 1. |
- Alm Rosenblad, Magnus, 1957, et al.
(author)
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Detection of signal recognition particle (SRP) RNAs in the nuclear ribosomal internal transcribed spacer 1 (ITS1) of three lineages of ectomycorrhizal fungi (Agaricomycetes, Basidiomycota)
- 2016
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In: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 13, s. 21-33
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Journal article (peer-reviewed)abstract
- During a routine scan for Signal Recognition Particle (SRP) RNAs in eukaryotic sequences, we surprisingly found in silico evidence in GenBank for a 265-base long SRP RNA sequence in the ITS1 region of a total of 11 fully identified species in three ectomycorrhizal genera of the Basidiomycota (Fungi): Astraeus, Russula, and Lactarius. To rule out sequence artifacts, one specimen from a species indicated to have the SRP RNA-containing ITS region in each of these genera was ordered and re-sequenced. Sequences identical to the corresponding GenBank entries were recovered, or in the case of a non-original but conspecific specimen differed by three bases, showing that these species indeed have an SRP RNA sequence incorporated into their ITS1 region. Other than the ribosomal genes, this is the first known case of non-coding RNAs in the eukaryotic ITS region, and it may assist in the examination of other types of insertions in fungal genomes.
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| 2. |
- Trona, Federica, et al.
(author)
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Neural coding merges sex and habitat chemosensory signals in an insect herbivore
- 2013
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In: Proceedings of the Royal Society of London. Biological Sciences. - : The Royal Society. - 0962-8452 .- 1471-2954. ; 280:1760, s. 20130267-
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Journal article (peer-reviewed)abstract
- Understanding the processing of odour mixtures is a focus in olfaction research. Through a neuroethological approach, we demonstrate that different odour types, sex and habitat cues are coded together in an insect herbivore. Stronger flight attraction of codling moth males, Cydia pomonella, to blends of female sex pheromone and plant odour, compared with single compounds, was corroborated by functional imaging of the olfactory centres in the insect brain, the antennal lobes (ALs). The macroglomerular complex (MGC) in the AL, which is dedicated to pheromone perception, showed an enhanced response to blends of pheromone and plant signals, whereas the response in glomeruli surrounding the MGC was suppressed. Intracellular recordings from AL projection neurons that transmit odour information to higher brain centres, confirmed this synergistic interaction in the MGC. These findings underscore that, in nature, sex pheromone and plant odours are perceived as an ensemble. That mating and habitat cues are coded as blends in the MGC of the AL highlights the dual role of plant signals in habitat selection and in premating sexual communication. It suggests that the MGC is a common target for sexual and natural selection in moths, facilitating ecological speciation.
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| 3. |
- Ding, Baojian, et al.
(author)
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Sequence variation determining stereochemistry of a delta-11 desaturase active in moth sex pheromone biosynthesis
- 2016
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In: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 1879-0240 .- 0965-1748. ; 74, s. 68-75
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Journal article (peer-reviewed)abstract
- A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution.
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| 4. |
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| 5. |
- Tavella, T A, et al.
(author)
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Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases.
- 2021
-
In: Advances in Protein Chemistry and Structural Biology. - : Elsevier. - 1876-1631. ; 124, s. 275-309
-
Research review (peer-reviewed)abstract
- The discovery and development of a new drug is a complex, time consuming and costly process that typically takes over 10 years and costs around 1 billion dollars from bench to market. This scenario makes the discovery of novel drugs targeting neglected tropical diseases (NTDs), which afflict in particular people in low-income countries, prohibitive. Despite the intensive use of High-Throughput Screening (HTS) in the past decades, the speed with which new drugs come to the market has remained constant, generating doubts about the efficacy of this approach. Here we review a few of the yeast-based high-throughput approaches that can work synergistically with parasite-based, in vitro, or in silico methods to identify and optimize novel antiparasitic compounds. These yeast-based methods range from HTP screens to identify novel hits against promising parasite kinase targets to the identification of potential antiparasitic kinase inhibitors extracted from databases of yeast chemical genetic screens.
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| 6. |
- Nachin, Laurence, 1971, et al.
(author)
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Heterodimer formation within universal stress protein classes revealed by an in silico and experimental approach.
- 2008
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In: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 380:2, s. 340-50
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Journal article (peer-reviewed)abstract
- Universal stress proteins (Usps) are found in all kingdoms of life and can be divided into four classes by phylogenic analysis. According to available structures, Usps exist as homodimers, and genetic studies show that their cellular assignments are extensive, including functions relating to stress resistance, carbon metabolism, cellular adhesion, motility, and bacterial virulence. We approached the question of how Usps can achieve such a variety of functions in a cell by using a new procedure for statistical analysis of multiple sequence alignments, based on physicochemically related values for each amino acid residue of Usp dimer interfaces. The results predicted that Usp proteins within a class may, in addition to forming homodimers, be able to form heterodimers. Using Escherichia coli Usps as model proteins, we confirmed the existence of such interactions. We especially focused on class I UspA and UspC and demonstrated that they are able to form homo- and heterodimers in vitro and in vivo. We suggest that this ability to form both homo- and heterodimers may allow for an expansion of the functional repertoire of Usps and explains why organisms usually contain multiple usp paralogues.
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| 7. |
- Larsson, Daniel, 1981-
(author)
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Exploring the Molecular Dynamics of Proteins and Viruses
- 2012
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Doctoral thesis (other academic/artistic)abstract
- Knowledge about structure and dynamics of the important biological macromolecules — proteins, nucleic acids, lipids and sugars — helps to understand their function. Atomic-resolution structures of macromolecules are routinely captured with X-ray crystallography and other techniques. In this thesis, simulations are used to explore the dynamics of the molecules beyond the static structures.Viruses are machines constructed from macromolecules. Crystal structures of them reveal little to no information about their genomes. In simulations of empty capsids, we observed a correlation between the spatial distribution of chloride ions in the solution and the position of RNA in crystals of satellite tobacco necrosis virus (STNV) and satellite tobacco mosaic virus (STMV). In this manner, structural features of the non-symmetric RNA could also be inferred.The capsid of STNV binds calcium ions on the icosahedral symmetry axes. The release of these ions controls the activation of the virus particle upon infection. Our simulations reproduced the swelling of the capsid upon removal of the ions and we quantified the water permeability of the capsid. The structure and dynamics of the expanded capsid suggest that the disassembly is initiated at the 3-fold symmetry axis.Several experimental methods require biomolecular samples to be injected into vacuum, such as mass-spectrometry and diffractive imaging of single particles. It is therefore important to understand how proteins and molecule-complexes respond to being aerosolized. In simulations we mimicked the dehydration process upon going from solution into the gas phase. We find that two important factors for structural stability of proteins are the temperature and the level of residual hydration. The simulations support experimental claims that membrane proteins can be protected by a lipid micelle and that a non-membrane protein could be stabilized in a reverse micelle in the gas phase. A water-layer around virus particles would impede the signal in diffractive experiments, but our calculations estimate that it should be possible to determine the orientation of the particle in individual images, which is a prerequisite for three-dimensional reconstruction.
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| 8. |
- Sahin, Cagla, et al.
(author)
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Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization
- 2022
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:27, s. 11949-11954
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Journal article (peer-reviewed)abstract
- α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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| 9. |
- Sridhara, Sagar, 1989
(author)
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Multiple structural flavors of RNase P in precursor tRNA processing.
- 2024
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In: Wiley interdisciplinary reviews. RNA. - 1757-7012. ; 15:2
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Journal article (peer-reviewed)abstract
- The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
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| 10. |
- Panagaki, Dimitra, et al.
(author)
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Nuclear envelope budding is a response to cellular stress.
- 2021
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 118:30
-
Journal article (peer-reviewed)abstract
- Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.
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| 11. |
- Hemsworth, Glyn R., et al.
(author)
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Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut
- 2016
-
In: Open Biology. - : Royal Society of London. - 2046-2441. ; 6:7
-
Journal article (peer-reviewed)abstract
- The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta) genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an alpha-xylosidase, a beta-glucosidase, and two alpha-L-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
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| 12. |
- Larsbrink, Johan, 1982, et al.
(author)
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A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin
- 2016
-
In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:260
-
Journal article (peer-reviewed)abstract
- Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown.ResultsHere we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm.ConclusionsBy combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.
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| 13. |
- Wu, Min, 1986, et al.
(author)
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Proline 411 biases the conformation of the intrinsically disordered plant UVR8 photoreceptor C27 domain altering the functional properties of the peptide
- 2019
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
-
Journal article (peer-reviewed)abstract
- UVR8 (UV RESISTANCE LOCUS 8) is a UV-B photoreceptor responsible for initiating UV-B signalling in plants. UVR8 is a homodimer in its signalling inactive form. Upon absorption of UV radiation, the protein monomerizes into its photoactivated state. In the monomeric form, UVR8 binds the E3 ubiquitin ligase COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), triggering subsequent UV-B-dependent photomorphogenic development in plants. Recent in vivo experiments have shown that the UVR8 C-terminal region (aa 397-423; UVR8(C27)) alone is sufficient to regulate the activity of COP1. In this work, CD spectroscopy and NMR experiments showed that the UVR8(C27) domain was non-structured but gained secondary structure at higher temperatures leading to increased order. Bias-exchange metadynamics simulations were also performed to evaluate the free energy landscape of UVR8(C27). An inverted free energy landscape was revealed, with a disordered structure in the global energy minimum. Flanking the global energy minimum, more structured states were found at higher energies. Furthermore, stabilization of the low energy disordered state was attributed to a proline residue, P411, as evident from P411A mutant data. P411 is also a key residue in UVR8 binding to COP1. UVR8(C27) is therefore structurally competent to function as a molecular switch for interaction of UVR8 with different binding partners since at higher free energies different structural conformations are being induced in this peptide. P411 has a key role for this function.
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| 14. |
- Zeng, Jiao, et al.
(author)
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High-resolution structure of a fish aquaporin reveals a novel extracellular fold
- 2022
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In: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
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Journal article (peer-reviewed)abstract
- Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (Anabas testudineus), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.
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| 15. |
- Purayil, Siju, et al.
(author)
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Neuropeptides in the Antennal Lobe of the Yellow Fever Mosquito, Aedes aegypti.
- 2014
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In: Journal of Comparative Neurology. - : Wiley. - 0021-9967 .- 1096-9861. ; 522, s. 592-608
-
Journal article (peer-reviewed)abstract
- For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592-608, 2014. (c) 2013 Wiley Periodicals, Inc.
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| 16. |
- D'Angiolo, M., et al.
(author)
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A yeast living ancestor reveals the origin of genomic introgressions
- 2020
-
In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 587, s. 420-425
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Journal article (peer-reviewed)abstract
- A yeast clonal descendant of an ancient hybridization event is identified and sheds light on the early evolution of the Saccharomyces cerevisiae Alpechin lineage and its abundant Saccharomyces paradoxus introgressions. Genome introgressions drive evolution across the animal(1), plant(2) and fungal(3) kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast(4), has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage(5), which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.
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| 17. |
- Stenlid, Jan
(author)
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The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes
- 2012
-
In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 336, s. 1715-1719
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Journal article (peer-reviewed)abstract
- Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.
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| 18. |
- Xu, Bo, 1980-
(author)
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Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
- 2014
-
Doctoral thesis (other academic/artistic)abstract
- The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions.The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization.A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals.Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours.In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.
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| 19. |
- Allison, Timothy M., et al.
(author)
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Complementing machine learning‐based structure predictions with native mass spectrometry
- 2022
-
In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6
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Journal article (peer-reviewed)abstract
- The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
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| 20. |
- Conti, Luca, et al.
(author)
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Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation
- 2016
-
In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Journal article (peer-reviewed)abstract
- Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.
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| 21. |
- Fröhlich, Christopher, et al.
(author)
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Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1.
- 2020
-
In: The Journal of antimicrobial chemotherapy. - : Oxford University Press (OUP). - 1460-2091 .- 0305-7453. ; 75:9, s. 2554-2563
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Journal article (peer-reviewed)abstract
- MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure.To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1.The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1).Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity.These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.
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| 22. |
- Khaji, Erfan, et al.
(author)
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3D protein structure prediction using Imperialist Competitive algorithm and half sphere exposure prediction
- 2016
-
In: Journal of Theoretical Biology. - : Elsevier BV. - 0022-5193. ; 391, s. 81-87
-
Journal article (peer-reviewed)abstract
- Predicting the native structure of proteins based on half-sphere exposure and contact numbers has been studied deeply within recent years. Online predictors of these vectors and secondary structures of amino acids sequences have made it possible to design a function for the folding process. By choosing variant structures and directs for each secondary structure, a random conformation can be generated, and a potential function can then be assigned. Minimizing the potential function utilizing meta-heuristic algorithms is the final step of finding the native structure of a given amino acid sequence. In this work, Imperialist Competitive algorithm was used in order to accelerate the process of minimization. Moreover, we applied an adaptive procedure to apply revolutionary changes. Finally, we considered a more accurate tool for prediction of secondary structure. The results of the computational experiments on standard benchmark show the superiority of the new algorithm over the previous methods with similar potential function. © 2015 Elsevier Ltd.
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| 23. |
- Landreh, Michael, et al.
(author)
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Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
- 2017
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Journal article (peer-reviewed)abstract
- Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
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| 24. |
- Papadopoulos, Evangelos, et al.
(author)
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Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli
- 2012
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In: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1824:12, s. 1401-1408
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Journal article (peer-reviewed)abstract
- The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K-a >= 10(17) M-1 at 23 degrees C, pH 7.0).
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| 25. |
- Reddy, Hemanth K.N., et al.
(author)
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Electron cryo-microscopy of bacteriophage PR772 reveals the elusive vertex complex and the capsid architecture
- 2019
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In: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Journal article (peer-reviewed)abstract
- Bacteriophage PR772, a member of the Tectiviridae family, has a 70 nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 angstrom, while the resolution of the protein capsid is 2.3 angstrom. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T = 25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.
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